Production of xylitol from glucose by a recombinant strain

ABSTRACT

The present invention relates to a recombinant microbial host for the production of xylitol, the recombinant microbial host containing a nucleic acid sequence encoding a NAD + -specific D-arabitol 4-oxidoreductase (EC 1.1.1.11) using D-arabitol as substrate and producing D-xylulose as product, and a nucleic acid sequence encoding a NADPH-specific xylitol dehydrogenase using D-xylulose as substrate and producing xylitol as product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of application Ser. No. 15/319,385 filed Dec. 16,2016, which in turn is a U.S. national stage application ofInternational Patent Application No. PCT/EP2015/063549, filed Jun. 17,2015, which claims priority to EP 14305934.3, filed Jun. 18, 2014. Thedisclosure of the prior applications is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of using genetically modifiedmicroorganisms for the manufacture of xylitol, and a method of preparinga genetically modified microorganism that is capable of converting inone step readily available carbon sources, such as D-glucose, intoxylitol.

BACKGROUND OF THE INVENTION

Xylitol is a polyalcohol or sugar alcohol (alditol) of formula(CHOH)₃(CH₂OH)₂, that has applications in hygiene and nutraceuticalformulations and products.

Xylitol is used as a diabetic sweetener which is roughly as sweet assucrose with 33% fewer calories. Unlike other natural or syntheticsweeteners, xylitol is actively beneficial for dental health by reducingcaries to a third in regular use and helpful to remineralization.

Xylitol is naturally found in low concentrations in the fibers of manyfruits and vegetables, and can be extracted from various berries, oats,and mushrooms, as well as fibrous material such as corn husks and sugarcane bagasse, and birch.

However, industrial production starts from xylan (a hemicellulose)extracted from hardwoods or corncobs, which is hydrolyzed into xyloseand catalytically hydrogenated into xylitol.

Purification of xylose and also xylitol presents therefore a significantproblem. A number of processes of this type are known. U.S. Pat. Nos.4,075,406 and 4,008,285 can be mentioned as examples.

The reduction of D-xylose into xylitol can also be achieved in amicrobiological process using either yeast strains isolated from nature(wild type strains) or genetically engineered strains.

However, obtaining the substrate, D-xylose, in a form suitable for yeastfermentation is a problem because inexpensive xylose sources such assulphite liquor from pulp and paper processes contain impurities whichinhibit yeast growth.

An attractive alternative method for the manufacture of xylitol isobtaining it by fermentation of a cheap and readily available substrate,such as D-glucose.

In the state of the art, there are some recombinant microorganismsdescribed able to produce xylitol in certain amounts during a one-stepfermentation of any common carbon sources other than D-xylose andD-xylulose.

These recombinant microorganisms, especially osmophilic yeasts, are forexample Zygosaccharomyees rouxii, Candida polymorpha, and Torulopsiscandida, initially known as producers of significant amounts of axylitol closely related pentitol, which is D-arabitol, from D-glucose(Lewis D. H. & Smith D. C., 1967, New Phytol. 66:143-184).

Thus, the international patent application WO 94/10325 provides methodsfor constructing such recombinant hosts being capable of producingxylitol when grown on carbon sources other than D-xylulose or D-xylose,and other than polymers or oligomers or mixtures thereof.

In the current patent application, this goal is achieved throughmodification of the metabolism of the desired microorganism, preferablya naturally occurring yeast microorganism, by introducing and expressingdesired heterologous genes.

This goal is also achieved by further modification of the metabolism ofsuch desired microorganism, so as to overexpress and/or inactivate theactivity or expression of certain genes homologous to such microorganismin its native state.

The method provided in this patent application for the production ofxylitol utilized an altered D-arabitol biosynthesis pathway, and suchpathway being notably altered by extending the preexisting D-arabitolpathway by the introduction and overexpression of the genes coding forD-xylulose-forming D-arabitol dehydrogenase (EC 1.1.1.11) and xylitoldehydrogenase (EC 1.1.1.9) into an D-arabitol-producing microorganism.

However, the yield of xylitol in the trials described in WO 94/10325 wasonly approximately 7.7 g/l after 48 hours of cultivation in a mediumwith yeast extract.

To try to optimize this first result, it was further proposed in WO94/10325 to inactivate, using mutagenesis or gene disruption, the genescoding for transketolase (EC 2.2.1.1) and/or the gene coding forD-xylulokinase (EC 2.7.1.17), and also to overexpress the genes codingfor the enzymes of the oxidative branch of the pentose-phosphatepathway, and specifically D-glucose-6-phosphate dehydrogenase (EC1.1.1.49) and/or 6-phospho-D-gluconate dehydrogenase (EC 1.1.1.44)and/or D-ribulose-5-phosphate epimerase gene (EC 5.1.3.1) in suchmicroorganisms.

But, whatever the genetic combination employed, the xylitol titer wasnever more than 9 g/l.

There is therefore still an unsatisfied need for a better geneticmanipulation of xylitol producing strains in order to optimize itsproduction, and thus make it commercially profitable.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant host cell capable ofproducing xylitol, wherein said host cell comprises:

an heterologous nucleic acid sequence encoding a NAD⁺-specificD-arabitol 4-oxidoreductase (EC 1.1.1.11) using D-arabitol as substrateand producing D-xylulose as product; and,

an heterologous nucleic acid sequence encoding a NADPH-specific xylitoldehydrogenase using D-xylulose as substrate and producing xylitol asproduct.

Preferably, the host cell does not consume D-arabitol as a sole carbonsource. More preferably, the host cell is selected from bacteria, fungiand yeast. In a preferred embodiment, the host cell is an osmophilic orosmotolerant yeast, in particular Pichia ohmeri.

Preferably, the NAD⁺-specific D-arabitol 4-oxidoreductase (EC 1.1.1.11)is from E. coli or Ralstonia solanacearum. More preferably, theNAD⁺-specific D-arabitol 4-oxidoreductase (EC 1.1.1.11) comprises orconsists in the sequence of SEQ ID No 2 or 43 or a sequence with 1-3additions, substitutions or deletions of amino acids. In a preferredembodiment, the sequence encoding the NAD⁺-specific D-arabitol4-oxidoreductase (EC 1.1.1.11) comprises or consists in the sequence ofSEQ ID No 3 or 42.

Preferably, the NADPH-specific xylitol dehydrogenase is a xylitoldehydrogenase from Pichia stipitis or Gluconobacter oxydans mutated forchanging the cofactor specificity from NADH to NADPH. More preferably,the NADPH-specific xylitol dehydrogenase comprises or consists in thesequence of SEQ ID No 5 or 8 or a sequence with 1-3 additions,substitutions or deletions of amino acids. In a preferred embodiment,the sequence encoding the NADPH-specific xylitol dehydrogenase comprisesor consists in the sequence of SEQ ID No 6 or 9.

Preferably, the host cell is capable of producing a xylitol titer of atleast 15 g/l in the supernatant after a 48 h culture.

Preferably, the host cell is a strain selected from strains I-4982,I-4960 and I-4981 deposited at the CNCM.

Preferably, the host cell comprises several copies of a sequenceencoding a NAD⁺-specific D-arabitol 4-oxidoreductase and/or severalcopies of a sequence encoding the NADPH-specific xylitol dehydrogenase.

The present invention also relates to a method for producing xylitolcomprising culturing a recombinant host cell as described above, andrecovering xylitol.

It additionally relates to a nucleic acid comprising or consisting in anucleic acid sequence selected from the group consisting of SEQ ID No 1,3, 7 and 9, an expression cassette or vector comprising said nucleicacid.

Finally, the present invention relates to the use of a recombinant hostcells according to the present invention for producing xylitol.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, by a “carbon source other than D-xylose and D-xylulose”is meant a carbon substrate for xylitol production other than D-xyloseand D-xylulose or polymers or oligomers or mixtures thereof (such asxylan and hemicellulose). The carbon source preferably includesD-glucose, and various D-glucose-containing syrups and mixtures ofD-glucose with other sugars.

As used herein, by “gene” is meant a nucleic acid sequence that may codefor a protein, in particular a DNA sequence.

As used herein, by “vector” is meant a plasmid or any other DNA sequencewhich is able to carry genetic information, specifically DNA, into ahost cell. The vector can further contain a marker or reporter suitablefor use in the identification of cells transformed with the vector, andorigins of replication that allow for the maintenance and replication ofthe vector in one or more prokaryotic or eukaryotic hosts. A “plasmid”is a vector, generally circular DNA that is maintained and replicatesautonomously in at least one host cell.

As used herein, by “expression vector” is meant a vector similar to avector but which supports expression of a gene or encoding nucleic acidthat has been cloned into it, after transformation into a host. Thecloned gene or encoding nucleic acid is usually placed under the controlof (i.e., operably linked to) certain control sequences such as promotersequences, that can be provided by the vector or by the recombinantconstruction of the cloned gene. Expression control sequences will varydepending on whether the vector is designed to express the operablylinked gene in a prokaryotic or eukaryotic host and can additionallycontain transcriptional elements such as enhancer elements (upstreamactivation sequences) and termination sequences, and/or translationalinitiation and termination sites.

As used herein, by “host” is meant a cell, prokaryotic or eukaryotic,that is utilized as the recipient and carrier of recombinant material.

As used herein, by “Oxidative Branch of the Pentose-Phosphate Pathway”is meant to include the part of the pentose-phosphate shunt thatcatalyzes oxidative reactions, such as reactions catalyzed byD-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gluconolactonase (EC3.1.1.17), and 6-phospho-D-gluconate dehydrogenase (EC 1.1.1.44), andthat utilizes hexose substrates to form pentose phosphates. The“non-oxidative” part of the pentose-phosphate pathway (which alsocatalyzes the net formation of ribose from D-glucose) is characterizedby non-oxidative isomerizations such as the reactions catalyzed bytransketolase (EC 2.2.1.1), ribose-5-phosphate isomerase (EC 5.3.1.6),D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1) and transaldolase (EC2.2.1.2). See Biological Chemistry, H. R. Mahler & E. H. Cordes, Harper& Row, publishers, New York, 1966, pp. 448-454.

As used herein, by “encoding nucleic acid” is meant a nucleic acidmolecule (preferably DNA). Encoding nucleic acid is capable of encodinga protein and can be prepared from a variety of sources. These sourcesinclude genomic DNA, cDNA, synthetic DNA, and combinations thereof.

“Heterologous”, as used herein, is understood to mean that a gene orencoding sequence has been introduced into the cell by geneticengineering. It can be present in episomal or chromosomal form. The geneor encoding sequence can originate from a source different from the hostcell in which it is introduced. However, it can also come from the samespecies as the host cell in which it is introduced but it is consideredheterologous due to its environment which is not natural. For example,the gene or encoding sequence is referred to as heterologous because itis under the control of a promoter which is not its natural promoter, itis introduced at a location which differs from its natural location. Thehost cell may contain an endogenous copy of the gene prior tointroduction of the heterologous gene or it may not contain anendogenous copy.

OBJECT OF THE INVENTION

According to the invention, the native metabolic pathways of a specificmicrobial host are manipulated so as to decrease or eliminate theutilization of carbon into purposes other than xylitol production.

Such a genetically modified host strain is thus able to produces xylitolin one fermentation step with a high yield. For instance, the xylitoltiter after 48 h of culture in the supernatant is more than 15 g/l,preferably more than 25 g/l, still more preferably more than 50, 60, 70,80, 90 or 100 g/l.

In the practical realization of the invention, the genetically modifiedhost of the invention is also characterized by its ability to synthesizexylitol from structurally unrelated carbon sources such as D-glucose,and not just from D-xylose and/or D-xylulose.

Preferably, the genetically modified host of the invention is alsocapable of secreting the synthesized xylitol into the medium.

Specifically, in the exemplified and preferred embodiments, thegenetically modified host of the invention is characterized by a pathwayin which arabitol is an intermediate in xylitol formation.

Accordingly, the recombinant host strain of the invention ischaracterized by the following genetic alterations:

(1) a heterologous nucleic acid encoding a protein possessingNAD⁺-specific D-arabitol 4-oxidoreductase (D-xylulose-forming) activityhas been introduced into the host cell thus providing for the conversionof D-arabitol to D-xylulose; and

(2) a heterologous nucleic acid encoding a protein possessingNADPH-specific xylitol dehydrogenase activity has been introduced intothe host cell—thus providing for the conversion of D-xylulose toxylitol.

The Choice of the Microorganism

The microorganisms or host strains suitable for the present inventionare capable of producing D-arabitol from glucose. More particularly,they are capable of producing significant amounts of D-arabitol fromglucose under high osmotic pressure medium.

By “high osmotic pressure medium” is intended here to refer to mediumcontaining 10-60% D-glucose, preferably about 25% D-glucose.

By “significant amounts of D-arabitol” is intended at least 100 g/L ofD-arabitol. In particular, a microorganism or host strain is consideredas producing significant amounts of D-arabitol when the microorganism orhost strain produces 100 g/L D-arabitol in a medium containing 25%D-glucose in batch conditions.

Examples of host strains capable of producing significant amounts ofD-arabitol from glucose include the osmophilic or osmotolerant yeasts,in particular those belonging to the species Pichia, Kodamaea, Candida,Zygoaccharomyces, Debaromyces, Metschnikowia and Hansenula; or theD-arabitol producing fungi, in particular those belonging to the speciesDendryphiella and Schizophyllum, in particular Dendryphiella salina andSchizophyllum commune.

Examples of the microorganisms of the genus Pichia include Pichiaohmeri, Pichia stipitis, Pichia farinosa, Pichia haplophila. Examples ofthe microorganisms of the genus Candida include Candida polymorpha andCandida tropicalis. Examples of the microorganisms of the genusZygoaccharomyces include Zygoaccharomyces rouxii. Other examples includeTorulopsis candida and Torulaspora hansenii. Examples of themicroorganisms of the genus Metschnikowia include Metschnikowiapulcherrima, Metschnikowia reukaufii, Metschnikowia bicuspidate,Metschnikowia lunate and Metschnikowia zobellii. As specific strains,Metschnikowia pulcherrima ATCC 18406, Metschnikowia reukaufii ATCC18407, Metschnikowia bicuspidate ATCC 24179, Metschnikowia lunala ATCC22033, Metschnikowia zobellii ATCC 22302 and Metschnikowia pulcherrimaFERM BP-7161 can be mentioned. These strains can be obtained fromAmerican Type Culture Collection, Address: 12301 Parklawn Drive,Rockville, Md. 20852, United States of America. Metschnikowiapulcherrima FERN BP-7161 was originally deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(postal code: 305-8566, 1-3 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan) on Jan. 16, 1998, under the deposition number of FERM P-16592 andtransferred from the original deposit to international deposit based onBudapest Treaty on May 15, 2000, and has been deposited as depositionnumber of FERM BP-7161. In a specific aspect, the microorganism has theaccession number FERM BP-7161. For more information, refer to EP1065276.

The microorganism can be genetically engineered in order to improve itscapacity of producing D-arabitol and/or reducing its capacity to useD-arabitol for a goal distinct from xylitol production.

For the invention, the host strain is advantageously chosen by itsspecific metabolic attributes:

-   -   it may be a producer of significant amounts of D-arabitol from        glucose as detailed above, in particular under high osmotic        pressure medium, for example medium containing 10-60% D-glucose,        and preferably 25% D-glucose (“Normal” medium usually contains        only 2-3% glucose.)    -   it may not consume D-arabitol as a sole carbon source;    -   its redox balance permits the generation of the cofactors needed        for the corresponding ketopentose/pentose alcohol conversion.

In one embodiment of the invention, the osmophilic yeast Pichia ohmeri(and their mutagenized derivatives) has been employed as a model and asa preferred host. Pichia ohmeri has initially been isolated fromcucumber brine and commonly used in food industry for fermentation inpickles, rinds, and fruits.

It is known by the one skilled in the art that yeasts species such asPichia, Zygosaccharomyces, Debaromyces and Hansenula are able to grow inlow water activity environments, on the opposite of Saccharomycescerevisiae. These osmotolerant or osmophilic yeasts accumulatecompatible solute like glycerol, D-arabitol, erythritol and mannitolwhich protect and stabilize enzymes, thereby enabling the cellularfunctions in osmotic conditions of growth. The polyols produced alsoplay a role in redox balancing.

In a preferred aspect, the microorganism is Pichia ohmeri. Indeed, themain characteristic of the host strain Pichia ohmeri is to produce onlyD-arabitol as compatible solute, in contrast to Zygosaccharomyces rouxiiproducing glycerol and D-arabitol. In addition, the metabolic pathwayfrom glucose to D-arabitol is well known in Pichia ohmeri.

As described in Zygoaccharomyces rouxii (J. M. INGRAM and W. A. WOOD,1965, Journal of Bacteriology, Vol. 89, N°5, 1186-1194), the carbon fluxin Pichia ohmeri goes through the oxidative part of thePentose-Phosphate Pathway (PPP) to convert D-Glucose into D-ribulose-5-Pwith the concomitant production of two molecules of NADPH.D-ribulose-5-P is dephosphorylated to D-ribulose and then reduced toD-arabitol. In Pichia ohmeri host strain, the Pentose Phosphate Pathway(PPP) is very active and has been determined to be higher than 50%.

In a preferred embodiment, the host cell is a mutant Pichia ohmerideposited on Mar. 7, 2012, with the Collection Nationale de Cultures deMicroorganismes [National Collection of Microorganism Cultures] of theInstitut Pasteur (CNCM). 25 rue du Docteur Roux, 75724 PARIS Cedex 15,under number 1-4605.

Redox Reactions and Enzymes

The cofactors NADH and NADPH are essential to a multitude of biologicalfunctions, acting in so-called redox reactions as carriers of electronsfrom one reaction to another. Cells need to maintain the metabolicequilibrium of the two redox couples NADH/NAD⁺ and NADPH/NADP⁺ knowingthat the NADPH/NADP⁺ couple is maintained in a more reduced state thanthe NADH/NAD⁺ couple to provide a thermodynamic driving force. NADH,which is mostly found in the oxidized form NAD⁺, is the main co-factorin catabolic reactions where it is involved in the oxidative release ofenergy from nutrients. In contrast to NADH, NADPH is re-oxidizedexclusively in anabolic reactions or during times of oxidative stress.

Any metabolic engineering strategy that involves redox reactions has tofunction under these cellular constraints. It has been done in thegenetically modified strain that is the object of the invention.

As found by the inventor, notably described in the PhD Thesis entitled“Contribution à l'étude du metabolisme des pentitols chez Pichia ohmeri”(Sophie Huchette, University of Sciences and Technics of Lille, 1992),it has been demonstrated that the reactions involved into theoxidoreduction of ketopentoses are catalyzed by two different enzymes.

Thus, the host strain has an enzyme defined as a NADPH-specificD-ketopentose-oxidoreductase, forming D-arabitol from D-ribulose andforming xylitol from D-xylulose. The host strain also possesses aNADH-specific D-ketopentose-oxidoreductase, forming ribitol and xylitolrespectively from D-ribulose and D-xylulose. This enzyme is closed tothe well-known NAD⁺-specific xylitol dehydrogenase E.C 1.1.1.9 fromPichia stipitis (XYL2). As only intracellular D-ribulose is available incontrast to D-xylulose, the host strain balances the NADPH/NADP⁺ redoxcouple directly with the re-oxidization of NADPH through cytosolicformation of D-arabitol from D-ribulose. Then, D-arabitol is secretedinto the broth via a passive diffusion.

The inventors found that the lack in intracellular D-xylulose would bethe main reason for the non-production of xylitol by the host straineven if Pichia ohmeri possesses all the enzymatic tools to produce thispolyol via NADH- or NADPH-specific-D-ketopentose-oxidoreductase.

Indeed, it was chosen to clone into the wild type host strain Pichiaohmeri a gene encoding a protein possessing NAD⁺-specific D-arabitol4-oxidoreductase (D-xylulose-forming) activity (E.C.1.1.1.11) allowingthe cytosolic D-arabitol to be converted to D-xylulose and NADH.

So, intracellular D-xylulose becomes available into the geneticallymodified strain and could be reduced by the intrinsic NADH- andNADPH-specific-D-ketopentose-oxidoreductase. However, the strain isdevoid of the endogenous enzymes able to efficiently transformD-xylulose into xylitol. Therefore, it is necessary to geneticallyengineer the strain in order to introduce a heterologous xylitoldehydrogenase.

In the patent WO 94/10325, it was chosen to clone the NAD⁺-specificXylitol dehydrogenase (E.C 1.1.1.9) from Pichia stipitis (XYL2) allowingthe production of xylitol and balancing the NADH/NAD⁺ redox couple withthe oxidation of NADH produced by the previous metabolic step. But asmentioned before, the results are not really convincing.

The inventors found that, by cloning a gene encoding a mutated proteinpossessing NADPH-specific xylitol dehydrogenase, the D-xylulose isconverted to xylitol to balance the NADPH/NADP⁺ redox couple such asdone by the intrinsic production of D-arabitol from D-ribulose.

Due to its low affinity of the NADPH-specificD-ketopentose-oxidoreductase for D-arabitol, the Pichia ohmeri wild typehost strain does not consume the extracellular D-arabitol.

Because of the introduction of a NAD⁺-specific D-arabitol4-oxidoreductase (D-xylulose-forming) activity into the geneticallymodified strain, the D-arabitol produced into the broth could be wellconsumed by the modified strain the same way as the cytosolicD-arabitol.

Consequently, xylitol is produced at the same time from intracellularand extracellular D-arabitol.

Its production could be improved by enhancing the efficiency of thexylitol pathway extension to totally avoid the exportation of theintermediary D-arabitol.

Thus only xylitol would be produced from D-glucose with the samephysiological effect as D-arabitol. This improvement could be the resultof the genetic modifications but also of the adaptation of the cultureconditions.

The Choice of the Two Enzymatic Activities to be Cloned in the HostStrain

The choice of these two enzymatic activities is supported by theircofactor specificity, as described above.

The first enzyme oxidizes D-arabitol into D-xylulose.

Two types of D-arabitol dehydrogenases are known: D-xylulose-forming (EC1.1.1.11) (D-arabinitol NAD⁺ 4-oxidoreductase) and D-ribulose-forming(EC 1.1.1.250). Unless otherwise stated, it is the D-xylulose-formingarabitol dehydrogenase that is intended herein and referred to herein asarabitol dehydrogenase. D-ribulose-forming dehydrogenases are found inwild-type yeasts and fungi.

D-xylulose-forming arabitol dehydrogenases are mainly known in bacteria.For instance, they have been identified in Enterobacteriaceae, inparticular E. coli, Klebsiella aerogenes, and Aerobacter aerogenesstrain PRL-R3, in Gluconobacter oxydans, and additionally also in Pichiastipitis. In particular, several enzymes are referenced in UniprotKBdatabase, such as, Klebsiella pneumoniae (# O052720), Ralstoniasolanacearum (# P58708), Yersinia pestis (# P58709), Aerobacteraerogenes (# L8BEF0), E. coli (# K3EX35, I2ZSJ5, W1BYD6, W1H8N7,E7U4R7).

For the purposes of the present invention, Escherichia coli is thepreferred source of the NAD⁺-specific D-arabitol 4-oxidoreductase(D-xylulose-forming) gene. More specifically, its amino acid sequence isdisclosed in SEQ ID No 2. In particular, SEQ ID Nos 1 and 3 disclosenucleic acids encoding NAD⁺-specific D-arabitol 4-oxidoreductase ofEscherichia coli. The encoding sequence has been optimized for Pichiaohmeri by taking into account its codon specificity.

In addition, Ralstonia solanacearum is also a preferred source of theNAD⁺-specific D-arabitol 4-oxidoreductase (D-xylulose-forming) gene.More specifically, its amino acid sequence is disclosed in SEQ ID No 43.In particular, SEQ ID No 42 disclose nucleic acids encodingNAD⁺-specific D-arabitol 4-oxidoreductase of Ralstonia solanacearum. Theencoding sequence has been optimized for Pichia ohmeri by taking intoaccount its codon specificity.

The second enzyme converts D-xylulose into xylitol.

Although the majority of yeasts and fungi possess an endogenous xylitoldehydrogenase (EC 1.1.1.9) gene, the change of their cofactorspecificity from NADH to NADPH is necessary for the implementation ofthe present invention. Indeed, a key aspect of the present invention isto use a NADPH-specific xylitol dehydrogenase. In addition, this enzymeis preferably overexpressed in the host.

Numerous xylitol dehydrogenases are known and several scientificarticles teach how to change the cofactor specificity from NADH toNADPH. Watanabe et al (J; Biol. Chem., 2005, 280, 10340-10345) disclosesmutated xylitol dehydrogenase of Pichia stipitis with a modifiedcofactor specificity, especially the triple mutant (D207A/I208R/F209S)and the quadruple mutant (D207A/I208R/F209S/N211R). The amino acidsequence of the quadruple mutant is disclosed in SEQ ID No 5. A doublemutant of xylitol dehydrogenase of Gluconobacter oxydans (D38S/M39R)with a NADPH cofactor specificity is disclosed in Ehrensberger et al(2006, Structure, 14, 567-575). The amino acid sequence of the doublemutant is disclosed in SEQ ID No 8.

The mutation and cloning of the Pichia stipitis XYL2 nucleic acidsequence encoding the NADPH-specific xylitol dehydrogenase have beenprepared by the inventors. In particular, SEQ ID Nos 4 and 6 disclosenucleic acids encoding specific NADPH xylitol dehydrogenase of Pichiastipitis.

Alternatively, the inventors have also performed the mutation andcloning of the Gluconobacter oxydans nucleic acid sequence encoding theNADPH-specific xylitol dehydrogenase. In particular, SEQ ID Nos 7 and 9disclose nucleic acids encoding NADPH specific xylitol dehydrogenase ofGluconobacter oxydans. The encoding sequence has been optimized forPichia ohmeri by taking into account its codon specificity.

Expression Cassette, Vector and Recombinant Host Cell

In a particular aspect, the present invention relates to a nucleic acidcomprising an encoding sequence optimized for Pichia ohmeri selectedfrom the group consisting of SEQ ID No 3, 7, 9 and 42.

It further relates to an expression cassette comprising a nucleic acidcomprising an encoding sequence optimized for Pichia ohmeri selectedfrom the group consisting of SEQ ID No 1, 3, 7, 9 and 42.

It also relates to the nucleic acid construct of SEQ ID No 4 and anucleic acid comprising said nucleic acid construct.

In addition, it relates to a recombinant vector, in particular anexpression vector, comprising said nucleic acid or expression cassette.Generally, an expression cassette comprises all the elements requiredfor gene transcription and translation into a protein. In particular, itcomprises a promoter, optionally an enhancer, a transcription terminatorand the elements for translation. More particularly, the promoter usedto control the expression of the NADPH-specific xylitol dehydrogenase isselected in order to drive a strong expression. Indeed, this enzyme ispreferably overexpressed in the host cell. Such promoters are well-knownin the art. For instance, the promoter could be the P. ohmeri ribulosereductase promoter (poRR) or the P. ohmeri phosphoglycerate kinase(poPGK1).

It relates to a recombinant vector, in particular an expression vector,comprising a nucleic acid encoding a NAD⁺-specific D-arabitol4-oxidoreductase and a nucleic acid encoding NADPH-specific xylitoldehydrogenase. It also relates to a kit comprising a recombinant vector,in particular an expression vector, comprising a nucleic acid encoding aNAD⁺-specific D-arabitol 4-oxidoreductase, and a recombinant vector, inparticular an expression vector, comprising a nucleic acid encodingNADPH-specific xylitol dehydrogenase.

Preferably, said NAD⁺-specific D-arabitol 4-oxidoreductase andNADPH-specific xylitol dehydrogenase are selected among the enzymesdisclosed above. In particular, said NAD⁺-specific D-arabitol4-oxidoreductase comprises or consists of an amino acid sequence of SEQID No 2 or 42 or a sequence with 1-3 additions, substitutions ordeletions of amino acids. In particular, said NADPH-specific xylitoldehydrogenase comprises or consists of an amino acid sequence of SEQ IDNo 5 or 8 or a sequence with 1-3 additions, substitutions or deletionsof amino acids.

A preferred vector is a plasmid. Suitable plasmids are well-known by theperson skilled in the art and can be for instance selected among thosespecifically disclosed in Examples.

Genetically modified host of the invention are first produced by cloningthe genes coding for NAD⁺-specific D-arabitol 4-oxidoreductase and forNADPH-specific xylitol dehydrogenase under control of suitable promotersinto a recombinant vector and introduced into the host cells of theD-arabitol producing organism by transformation.

The present invention relates to a recombinant or geneticallyengineering host cell comprising an heterologous nucleic acid sequenceencoding a NAD specific D-arabitol 4-oxidoreductase (EC 1.1.1.11) and anheterologous nucleic acid sequence encoding a NADPH-specific xylitoldehydrogenase. The NAD⁺-specific D-arabitol 4-oxidoreductase usesD-arabitol as substrate and produces D-xylulose as product. TheNADPH-specific xylitol dehydrogenase uses D-xylulose as substrate andproduces xylitol. The sequence encoding NADPH-specific xylitoldehydrogenase and NAD⁺-specific D-arabitol 4-oxidoreductase can beepisomal or be integrated into the chromosome of the host cell. Indeed,genetically stable transformants are preferably constructed throughtransformation systems using a vector, whereby a desired DNA isintegrated into the host chromosome. Such integration occurs de novowithin the cell or can be assisted by transformation with a vector whichfunctionally inserts itself into the host chromosome, with DNA elementswhich promote integration of DNA sequences in chromosomes.

The recombinant or genetically engineering host cell can compriseseveral copies of a sequence encoding a NAD⁺-specific D-arabitol4-oxidoreductase and/or several copies of a sequence encoding theNADPH-specific xylitol dehydrogenase, preferably integrated into thehost cell chromosome. In particular, the recombinant or geneticallyengineering host cell can comprise two, three or four sequences encodinga NAD⁺-specific D-arabitol 4-oxidoreductase and/or two, three or foursequences encoding the NADPH-specific xylitol dehydrogenase. Forinstance, the host cell may comprise two or three NAD⁺-specificD-arabitol 4-oxidoreductases from E. coli and/or one or twoNAD⁺-specific D-arabitol 4-oxidoreductases from R. solanacearum, morespecifically two or three NAD⁺-specific D-arabitol 4-oxidoreductasesfrom E. coli and/or one NAD⁺-specific D-arabitol 4-oxidoreductase fromR. solanacearum. The NAD⁺-specific D-arabitol 4-oxidoreductases can befrom the same organism or from different organisms. The NADPH-specificxylitol dehydrogenases can be from the same organism or from differentorganisms. For instance, the host cell may comprise one, two or threeNADPH-specific xylitol dehydrogenases from P. stipitis and/or one, twoor three NADPH-specific xylitol dehydrogenases from G. oxydans, morespecifically one NADPH-specific xylitol dehydrogenase from P. stipitisand/or three NADPH-specific xylitol dehydrogenases from G. oxydans.

In a particular aspect of the invention, the recombinant or geneticallyengineering host cell is a Pichia ohmeri strain comprising:

-   -   two NAD⁺-specific D-arabitol 4-oxidoreductases and two        NADPH-specific xylitol dehydrogenases; or    -   two NAD⁺-specific D-arabitol 4-oxidoreductases from E. coli and        two NADPH-specific xylitol dehydrogenases, one from P. stipitis        and the other from G. oxydans; or    -   two NAD⁺-specific D-arabitol 4-oxidoreductases and three        NADPH-specific xylitol dehydrogenases; or    -   two NAD⁺-specific D-arabitol 4-oxidoreductases from E. coli and        three NADPH-specific xylitol dehydrogenases, one from P.        stipitis and two from G. oxydans; or    -   three NAD⁺-specific D-arabitol 4-oxidoreductases and three        NADPH-specific xylitol dehydrogenases; or    -   three NAD⁺-specific D-arabitol 4-oxidoreductases, two from E.        coli and one from R. solanacearum, and three NADPH-specific        xylitol dehydrogenases, one from P. stipitis and two from G.        oxydans; or    -   four NAD⁺-specific D-arabitol 4-oxidoreductases and four        NADPH-specific xylitol dehydrogenases; or    -   four NAD⁺-specific D-arabitol 4-oxidoreductases, three from E.        coli and one from R. solanacearum, and four NADPH-specific        xylitol dehydrogenases, one from P. stipitis and three from G.        oxydans.

The host cell is selected among the microorganisms detailed above. In apreferred embodiment, the host cell is Pichia ohmeri. The starting hostcell is preferably the mutant Pichia ohmeri deposited at the CNCM undernumber I-4605.

In a particular aspect of the invention, the host cell is a strainselected from strains I-4982, I-4960 and I-4981 deposited at the CNCM.

The present invention relates to a method for producing xylitolcomprising culturing the recombinant or genetically engineering hostcell in a culture medium and recovering the produced xylitol.Preferably, the culture medium provides the microorganism with theconvenient carbon source. The carbon source preferably includesD-glucose, and various D-glucose-containing syrups and mixtures ofD-glucose with other sugars. The method may further comprises a step ofpurifying xylitol.

The present invention relates to the use of a recombinant or geneticallyengineering host cell as disclosed herein for producing xylitol.

Xylitol produced by such genetically modified strains can be purifiedfrom the medium of the hosts of the invention according to any techniqueknown in the art. For example, U.S. Pat. No. 5,081,026, incorporatedherein by reference, described the chromatographic separation of xylitolfrom yeast cultures. Thus, from the fermentation step, xylitol can bepurified from the culture medium using chromatographic steps asdescribed in U.S. Pat. No. 5,081,026, followed by crystallization.

Other characteristic features and advantages of the invention will beapparent on reading the following Examples. However, they are given hereonly as an illustration and are not limiting.

FIGURES AND SEQUENCES

FIG. 1: 12 ABYWMP: Restriction map of the synthesized NAD⁺-specificD-arabitol 4-oxidoreductase from E. coli flanked by AscI and SphIrestriction sites.

FIG. 2A: lig7.78: Restriction map of the NADH-specific xylitoldehydrogenase from Pichia stipitis.

FIG. 2B: 12AALQTP: Restriction map of the synthesized NADPH-specificxylitol dehydrogenase from Pichia stipitis flanked by Hindlll and SacIIrestriction sites.

FIG. 3: 13AAYSYP: Restriction map of the synthesized NADPH-specificxylitol dehydrogenase from Gluconobacter oxydans flanked by AscI andSphI restriction sites.

FIG. 4: Construction of an expression cassette consisting of an openreading frame flanked by a poRR promoter and terminator using overlapPCR.

FIG. 5: 12 AAMCJP: Restriction map of the synthesizedtagatose-3-epimerase of Pseudomonas cichorii flanked by Hindlll andSacII restriction sites.

FIG. 6: Construction of P. ohmeri shuttle vectors with poLEU2 and poURA3selection markers.

FIG. 7: pEVE2523: Restriction map of the P. ohmeri poURA3 expressionvector pEVE2523, with a cloned expression cassette containing the openreading frame of tagatose-3-epimerase of Pseudomonas cichorii flanked bya P. ohmeri ribulose reductase (poRR) promoter and terminator.

FIG. 8: pEVE2560: Restriction map of the P. ohmeri poLEU2 expressionvector pEVE2560, with a cloned expression cassette containing the openreading frame of tagatose-3-epimerase of Pseudomonas cichorii flanked bya P. ohmeri ribulose reductase (poRR) promoter and terminator.

FIG. 9: Construction of a P. ohmeri vector for overexpression ofGluconobacter oxydans NADPH-specific xylitol dehydrogenase.

FIG. 10: pEVE3284: Restriction map of the P. ohmeri pEVE3284 expressionvector, with a cloned expression cassette containing the NADPH-specificxylitol dehydrogenase of Gluconobacter oxydens flanked by a P. ohmeriribulose reductase (poRR) promoter and terminator.

FIG. 11: Construction of a P. ohmeri vectors for overexpression ofPichia stipitis NADPH-specific xylitol dehydrogenase.

FIG. 12: pEVE2562/pEVE2564: Restriction map of the P. ohmeripEVE2562/pEVE2564 expression vectors, with a cloned expression cassettecontaining the NADPH-specific xylitol dehydrogenase of Pichia stipitisflanked by a P. ohmeri ribulose reductase (poRR) promoter and terminatorwith either a poURA3 or poLEU2 selection marker, respectively.

FIG. 13: Construction of a P. ohmeri vector for overexpression of Pichiastipitis NADH-specific xylitol dehydrogenase.

FIG. 14: pEVE2563: Restriction map of the P. ohmeri pEVE2563 expressionvector, with a cloned expression cassette containing the NADH-specificxylitol dehydrogenase of Pichia stipitis flanked by a P. ohmeri ribulosereductase (poRR) promoter and terminator.

FIG. 15: Construction of a P. ohmeri vector for overexpression of E.coli NAD⁺-specific D-arabitol 4-oxidoreductase under the control of theP. ohmeri ribulose reductase (poRR) promoter and terminator using apoURA3 selection marker.

FIG. 16: pEVE2839: Restriction map of the P. ohmeri pEVE2839 expressionvector, with a cloned expression cassette containing the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli flanked by a P. ohmeri ribulosereductase (poRR) promoter and terminator.

FIG. 17: Construction of a P. ohmeri vector for overexpression of E.coli NAD⁺-specific D-arabitol 4-oxidoreductase under the control of theP. ohmeri phophoglycerate kinase (poPGK1) promoter and transketolase(poTKL) terminator using a poURA3 selection marker.

FIG. 18: pEVE3102: Restriction map of the P. ohmeri pEVE3102 expressionvector, with a cloned expression cassette containing the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli flanked by a P. ohmeriphosphoglycerate kinase (poPGK1) promoter and ribulose reductase (poRR)terminator.

FIG. 19: pEVE3123: Restriction map of the P. ohmeri pEVE3123 expressionvector, with a cloned expression cassette containing the NAD⁺-specificD-arabitol oxidoreductase of E. coli flanked by a P. ohmeriphosphoglycerate kinase (poPGK1) promoter and a transketolase (poTKL)terminator and a poURA3 selection marker.

FIG. 20: Construction of a P. ohmeri vector for overexpression of E.coli NAD⁺-specific D-arabitol 4-oxidoreductase under the control of theP. ohmeri phosphoglycerate kinase (poPGK1) promoter and transketolase(poTKL) terminator using a poLEU2 selection marker.

FIG. 21: pEVE3157: Restriction map of the P. ohmeri pEVE3157 expressionvector, with a cloned expression cassette containing the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli flanked by a P. ohmeriphosphoglycerate kinase (poPGK1) promoter and a transketolase (poTKL)terminator and a poLEU2 selection marker.

FIG. 22: Construction of a P. ohmeri loxP vector with a poLEU2 selectionmarker

FIG. 23: pEVE2787: Restriction map of the P. ohmeri pEVE2787 integrationvector, with a cloned P. ohmeri LEU2 selection marker under the controlof the endogenous promoter and terminator, flanked by two loxP sites.

FIG. 24: 12ABTV4P: Restriction map of the synthesized nat1 gene fromStreptomyces noursei flanked by AscI and SphI restriction sites.

FIG. 25: pEVE2798: Restriction map of the P. ohmeri pEVE2798 expressionvector, with a cloned nat1 marker under the control of a ribulosereductase (poRR) promoter and an orotidine-5′-phosphate decarboxylase(poURA3) terminator.

FIG. 26: Construction of a P. ohmeri loxP vector with a nat1 selectionmarker.

FIG. 27: pEVE2852: Restriction map of the P. ohmeri pEVE2852 integrationvector, with a cloned with a cloned nat1 marker under the control of aribulose reductase (poRR) promoter and an orotidine-5′-phosphatedecarboxylase (poURA3) terminator, flanked by two loxP sites.

FIG. 28: pEVE2855: Restriction map of the P. ohmeri pEVE2855 integrationvector, with a cloned fragment homologous to the 5′ region upstream ofthe LEU2 open reading frame and a nat1 selection marker flanked by twoloxP sites.

FIG. 29: Construction of a P. ohmeri loxP vector for the deletion of theLEU2 open reading frame.

FIG. 30: pEVE2864: Restriction map of the P. ohmeri pEVE2864 integrationvector, with a cloned fragment homologous to the 5′ region upstream ofthe LEU2 open reading frame and fragment homologous to the 3′ regiondownstream of the LEU2 open reading frame, and a nat1 selection markerflanked by two loxP sites.

FIG. 31: Construction of a double expression plasmids comprising theNADPH-specific xylitol dehydrogenase of P. stipitis and theNAD⁺-specific D-arabitol 4-oxidoreductase of E. coli.

FIG. 32: pEVE3318: Restriction map of the P. ohmeri pEVE3318 expressionvector, containing the double expression construct of the NADPH-specificxylitol dehydrogenase of P. stipitis and the NAD⁺-specific D-arabitol4-oxidoreductase of E. coli.

FIG. 33: pEVE2862: Restriction map of the P. ohmeri pEVE2862 expressionvector, containing the P. ohmeri LEU2 marker flanked by a P. ohmeriribulose reductase (poRR) promoter and an orotidine-6-phosphatedecarboxylase (poURA3) terminator.

FIG. 34: Construction of an integrative vector for the genomicexpression of the E. coli NAD+-specific D-arabitol 4-oxidoreductase geneand the P. stipitis NADPH-specific xylitol dehydrogenase gene in P.ohmeri.

FIG. 35: pEVE2865: Restriction map of the P. ohmeri pEVE2865 integrationvector, containing the P. ohmeri LEU2 marker flanked by two loxP sites.

FIG. 36: pEVE3387: Restriction map of the P. ohmeri pEVE3387 integrationvector, containing the double expression construct of the NADPH-specificxylitol dehydrogenase gene of P. stipitis and the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli with a P. ohmeri LEU2 selectionmarker flanked by two loxP sites.

FIG. 37: Construction of double/triple expression plasmids comprisingthe NADPH-specific xylitol dehydrogenase of G. oxydans and theNAD⁺-specific D-arabitol 4-oxidoreductase of E. coli.

FIG. 38: pEVE3322/pEVE3324: Restriction map of the P. ohmeripEVE3322/pEVE3324 expression vectors, containing either the doubleexpression construct of the NADPH-specific xylitol dehydrogenase of G.oxydans and the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli orthe triple expression construct of two NADPH-specific xylitoldehydrogenase genes of G. oxydans and one NAD⁺-specific D-arabitol4-oxidoreductase of E. coli.

FIG. 39: Construction of an integrative vector for the genomicexpression of the E. coli NAD+-specific D-arabitol 4-oxidoreductase geneand the G. oxydans NADPH-specific xylitol dehydrogenase gene in P.ohmeri.

FIG. 40: pEVE3390IpEVE3392: Restriction map of the P. ohmeripEVE3390/pEVE3392 integration vectors, containing either the doubleexpression construct of the NADPH-specific xylitol dehydrogenase of G.oxydans and the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli orthe triple expression construct of two NADPH-specific xylitoldehydrogenase genes of G. oxydans and one NAD⁺-specific D-arabitol4-oxidoreductase of E. coli with a P. ohmeri LEU2 selection markerflanked by two loxP sites.

FIG. 41: Construction of an integrative vector for the genomicexpression of the E. coli NAD+-specific D-arabitol 4-oxidoreductase geneand the G. oxydans NADPH-specific xylitol dehydrogenase gene in P.ohmeri.

FIG. 42: pEVE4390: Restriction map of the P. ohmeri pEVE4390 expressionvector, containing the double expression construct of the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli and the NADPH-specific xylitoldehydrogenase gene of G. oxydans with a P. ohmeri LEU2 selection markerflanked by two loxP sites.

FIG. 43: 13AB2EGF: Restriction map of the synthesized NAD+-specificD-arabitol 4-oxidoreductase from R. solanacearum flanked by AscI andSphI restriction sites.

FIG. 44: Construction of an integrative vector for the genomicexpression of the R. solanacearum NAD+-specific D-arabitol4-oxidoreductase gene and the G. oxydans NADPH-specific xylitoldehydrogenase gene in P. ohmeri

FIG. 45: pEVE3898: Restriction map of the P. ohmeri pEVE3898 expressionvector, with a cloned expression cassette containing the NAD+-specificD-arabitol 4-oxidoreductase of Ralstonia solanacearum flanked by a P.ohmeri ribulose reductase (poRR) promoter and terminator.

FIG. 46: pEVE4077: Restriction map of the P. ohmeri pEVE4077 expressionvector, with a double expression construct of the NADPH-specific xylitoldehydrogenase of G. oxydans and the NAD⁺-specific D-arabitol4-oxidoreductase of R. solanacearum.

FIG. 47: pEVE4377: Restriction map of the P. ohmeri pEVE4377 integrationvector, with a double expression construct of the NADPH-specific xylitoldehydrogenase of G. oxydans and the NAD⁺-specific D-arabitol4-oxidoreductase of R. solanacearum and the poLEU2 selection markerflanked by two loxP sites.

SEQUENCE LISTING

SEQ ID No Description 1 Sequence encoding NAD⁺-specific D-arabitol4-oxidoreductase from E. coli flanked by AscI and SphI restriction sites2 Amino acid sequence of NAD⁺-specific D-arabitol 4-oxidoreductase fromE. coli 3 Sequence encoding NAD⁺-specific D-arabitol 4-oxidoreductasefrom E. coli 4 Sequence encoding NADPH-specific xylitol dehydrogenasefrom Pichia stipitis flanked by HindIII and SacII restriction sites 5Amino acid sequence of NADPH-specific xylitol dehydrogenase from P.stipitis 6 Sequence encoding NADPH-specific xylitol dehydrogenase fromPichia stipitis 7 Sequence encoding NADPH-specific xylitol dehydrogenasefrom Gluconobacter oxydans flanked by AscI and SphI restriction sites 8Amino acid sequence of NADPH-specific xylitol dehydrogenase fromGluconobacter oxydans 9 Sequence encoding NADPH-specific xylitoldehydrogenase from Gluconobacter oxydans 10 Sequence encodingtagatose-3-epimerase of Pseudomonas cichorii ST24 11 Amino acid sequenceof tagatose-3-epimerase of Pseudomonas cichorii ST24 28 Sequenceencoding the nat1 gene of Streptomyces noursei flanked by AscI and SphIrestriction sites 42 Sequence encoding the NAD+-specific D-arabitol4-oxidoreductase from R. solanacearum flanked by AscI and SphIrestriction sites 43 Amino acid sequence of NAD+-specific D-arabitol4-oxidoreductase from R. solanacearum

EXAMPLES Example 1. Choice of a Pichia ohmeri Strain as Preferred Hostfor Genetic Engineering

As host strain of choice, Pichia ohmeri:

-   -   is a producer of significant amounts of arabitol from glucose,        under high osmotic pressure medium, for example medium        containing 10-60% D-glucose, and preferably 25% D-glucose        (“Normal” medium usually contains only 2-3% glucose.)    -   has a redox balance that permits the generation of the cofactors        needed.

As an illustration of its performances, the following tables indicatethe enzyme activities involved in the arabitol metabolic pathway ofPichia ohmeri (Sophie HUCHETTE Thesis, 1992)

The Hexose Monophosphate Pathway: from Glucose-6-P to D-Ribulose-5-P andD-Xylulose-5-P

The oxidative part of the PPP, also named the Hexose MonophosphatePathway (HMP), is a NADPH-producing pathway. The two NADP⁺-dependentenzymes which are Glucose-6-P dehydrogenase (E.C.1.1.1.49) and6-P-Gluconate dehydrogenase (E.C.1.1.1.44) participate to the oxidationof 1 mole of glucose-6-P in 1 mole of D-ribulose-5-P and generate 2moles of NADPH.

TABLE 1 Hexose Monophosphate Pathway in P. ohmeri ATCC 20209 EnzymesSpecific activity U/mg NADP⁺ G6P dehydrogenase 1.5 NADP⁺ 6PGdehydrogenase 0.55 One unit of enzyme activity was defined as theconsumption of 1 μmole of NAD(P)H or NAD(P)+ per minute per mL of crudeextract. One unit of specific activity was defined as one unit of enzymeactivity per mg of proteins in crude extract.

The kinetic parameters of the following enzymes were determined:D-ribulose-5-P 3-epimerase (E.C 5.1.3.1), D-ribose-5-P keto-isomerase(E.C.5.3.1.6), transketolase (E.C.2.2.1.1) and acidic phosphatases (E.C.3.1.3.2).

TABLE 2 Kinetic parameters of enzymes using D-Ribulose-5-P as substratein P. ohmeri ATCC 20209 Enzymes K_(M) mM V_(M) U/mg D-Ribulose-5-P3-epimerase 6.3 3 D-Ribose-5-P keto-isomerase 0.35 1.8 Acid phosphatase4.3 0.65 One unit of enzyme activity was defined as the consumption of 1μmole of NAD(P)H or NAD(P)+ per minute per mL of crude extract. One unitof specific activity was defined as one unit of enzyme activity per mgof proteins in crude extract.

TABLE 3 Kinetic parameters of enzymes using D-Xylulose-5-P as substratein P. ohmeri ATCC 20209 Enzymes K_(M) mM V_(M) U/mg D-Ribulose-5-P3-epimerase 6.6 0.7 Transketolase (D-ribose-5-P) 0.2 0.9 Transketolase(Erythrose-4-P) 0.6 1.45 Acid phosphatase 16 0.11 One unit of enzymeactivity was defined as the consumption of 1 μmole of NAD(P)H or NAD(P)+per minute per mL of crude extract. One unit of specific activity wasdefined as one unit of enzyme activity per mg of proteins in crudeextract.

In vivo, D-xylulose-5-P, synthetized from the epimerization ofD-ribulose-5-P, enters efficiently into the non-oxidative part of thePPP via the transketolization. Consequently, D-xylulose-5-P is notavailable for its dephosphorylation into D-xylulose.

NADH and NADPH Specific D-Ketopentose-oxidoreductases

D-Ribulose and D-Xylulose are produced by dephosphorylization ofD-Ribulose-5-P and D-Xylulose-5-P.

The Michaelis-Menten constants highlight the affinities of the NADH andNADPH-D-ketopentose-oxidoreductases for each substrate and thecorresponding maximum velocities.

TABLE 4 NADH-specific D-ketopentose-oxidoreductase kinetic parameters ofP. ohmeri ATCC 20209 Substrate K_(M) mM V_(M) U/mg D-Ribulose 90 1Ribitol 16 0.16 D-Xylulose 5 0.6 Xylitol 7 0.2 One unit of enzymeactivity was defined as the consumption of 1 μmole of NAD(P)H or NAD(P)+per minute per mL of crude extract. One unit of specific activity wasdefined as one unit of enzyme activity per mg of proteins in crudeextract.

NADH-specific D-ketopentose-oxidoreductase, forming ribitol and xylitolrespectively from D-ribulose and D-xylulose shows a higher affinity forD-xylulose than D-ribulose. The reverse reaction shows a good affinityfor xylitol and ribitol explaining the good growth of the host strain onthese two polyols.

TABLE 5 NADPH-specific D-ketopentose-oxidoreductase kinetic parametersof P. ohmeri ATCC 20209 Substrate K_(M) mM V_(M) U/mg D-Ribulose 72 3.4D-Arabitol 1300 0.8 D-Xylulose 262 1.5 Xylitol 200 0.15 One unit ofenzyme activity was defined as the consumption of 1 μmole of NAD(P)H orNAD(P)+ per minute per mL of crude extract. One unit of specificactivity was defined as one unit of enzyme activity per mg of proteinsin crude extract.

NADPH-specific D-ketopentose-oxidoreductase, forming D-arabitol fromD-ribulose and forming xylitol from D-xylulose shows a higher affinityfor D-ribulose than D-xylulose. The reverse reaction shows a very lowaffinity for D-arabitol explaining the non-growth of the host strain onthis polyol.

The two ketopentose-oxidoreductases from the host strain werecharacterized as different from the previous enzymes described inSaccharomyces rouxii by Ingram and Wood, 1965 (Journal of Bacteriology,vol. 89, n°5, 1186-1194). Indeed, in Saccharomyces rouxii, no forwardreaction was detected on D-ribulose and NADH and a backward reaction wasdetected on D-arabitol with NADPH.

The Haldane relationship predicts in vivo enzyme kinetic behaviors.

TABLE 6 Substrat/Product K_(eq) mM⁻¹ Haldane constants determination:NADH-specificD-Ketopentose-oxidoreductase D-Ribulose/Ribitol 78D-Xylulose/Xylitol 104 Haldane constants determination:NADPH-specificD-Ketopentose-oxidoreductase D-Ribulose/D-Arabitol 104D-Xylulose/Xylitol 24

The two enzymes favor the forward reaction (D-ketopentose oxidation)over the backward reaction (pentitol reduction).

The PPP in the host strain is extremely efficient and 2 moles of NADPHare generated from 1 mole of glucose consumed. Consequently, NADPH wouldbe available in excess for both anabolic reactions and maintenancereactions. The host strain must produce D-arabitol from D-ribulose orxylitol from D-xylulose to balance the NADPH/NADP⁺ redox couple.

The inhibitory effect of NADP⁺ on NADPH-specificD-ketopentose-oxidoreductase has been determined in vitro. The activityis 80% less when NADP⁺ is added in excess. Even if this concentration isnot compatible with the intracellular NADP⁺ concentration, this resultgives some overview of the role of the NADPH-specific D-ketopentoseoxidoreductase into the balance of the NADPH/NADP⁺ redox couple.

The host strain produces only D-arabitol from D-ribulose as D-xyluloseis not available because of the entrance of D-xylulose-5-P into thenon-oxidative part of the PPP. The link between the production ofD-arabitol and the NADPH/NADP⁺ redox balance has been demonstrated inthe host strain by evaluating the impact of the overexpression ofGlucose-6-P dehydrogenase onto the D-arabitol production. So, theobtained strain harbors a G6PDH activity 1.5 times higher and produces10% more of D-arabitol compared to the host strain (FR2772788).

Example 2. Pichia ohmeri Codon Usage

The codon usage of P. ohmeri was determined from the available DNA andcorresponding amino acid sequence of five P. ohmeri genes:transketolase, glucose-6-phosphate dehydrogenase (FR 2772788), ribulosereductase, beta-isopropylmalate dehydrogenase—LEU2 (Piredda andGaillardin, Yeast, vol. 10:1601-1612 (1994) and orotidine-5′-phosphatedecarboxylase—URA3 (Piredda and Gaillardin, 1994, supra).

Every individual gene was divided in nucleotide triplets encoding for asingle amino acid. The five genes consisted of a total of 2091 codons.

For each amino acid, the number of every codon present in the five geneswas counted, divided by 2091 and multiplied by 1000. This way, thefrequency of a specific codon in 1000 codons was estimated.

The preliminary codon usage of P. ohmeri is depicted in Table 7.

All heterologous genes expressed in P. ohmeri, except the xylitoldehydrogenase from P. stipitis, were codon optimized using this tableand the Optimizer program (Nucleic Acids Research, 2007, 35, W126-W131).

The obtained sequence was sent for gene synthesis after manual additionof recognition sites for restriction enzymes at the respective 5′ and 3′ends of the sequence encoding the enzyme.

TABLE 7 Codon usage table of P. ohmeri derived from 5 coding sequences(CDS) Pichia ohmeri [gbpln]: 5 CDS's (2091 codons) fields: [triplet][frequency: per thousand] ([number]) TTT 10.5(  22) TCT 30.6( 64) TTC30.1(  63) TCC 23.4( 49) TTA 5.3(  11) TCA 4.8( 10) TTG 64.1( 134) TCG9.6( 20) CTT 10.5(  22) CCT 12.0( 25) CTC 12.0(  25) CCC 0.0(  0) CTA0.0(  0) CCA 34.0( 71) CTG 2.9(  6) CCG 0.5(  1) ATT 27.7(  58) ACT22.0( 46) ATC 30.6(  64) ACC 24.4( 51) ATA 2.4(  5) ACA 3.3(  7) ATG14.3(  30) ACG 1.4(  3) GTT 27.3(  57) GCT 46.9( 98) GTC 19.1(  40) GCC27.7( 58) GTA 1.9(  4) GCA 11.0( 23) GTG 21.5(  45) GCG 3.3(  7) TAT7.7(  16) TGT 5.7(  12) TAC 27.3(  57) TGC 1.4(  3) TAA 1.4(  3) TGA0.0(  0) TAG 1.0(  2) TGG 12.9(  27) CAT 3.3(  7) CGT 5.7(  12) CAC15.8(  33) CGC 1.0(  2) CAA 12.4(  26) CGA 0.0(  0) CAG 17.7(  37) CGG0.5(  1) AAT 7.7(  16) AGT 1.9(  4) AAC 29.2(  61) AGC 2.4(  5) AAA11.0(  23) AGA 26.3(  55) AAG 64.1( 134) AGG 0.0(  0) GAT 23.0(  48) GGT60.7( 127) GAC 35.9(  75) GGC 10.5(  22) GAA 18.7(  39) GGA 12.0(  25)GAG 46.9(  98) GGG 1.0(  2)

Example 3. Cloning of the E. coli Bacterial NAD⁺-Specific D-Arabitol4-Oxidoreductase (D-Xylulose-Forming) Gene

A DNA fragment encoding the NAD⁺-specific D-arabitol 4-oxidoreductasealtD from E. coli was chemically synthesized (GeneArt® Gene Synthesis,Life Technologies, Regensburg, Germany), according to the submittedsequence of SEQ ID NO: 1.

Nucleotides 1441 to 2808 of sequence AF378082A (obtained from the NCBIGenBank database) coding for the altD gene were used as template andsubjected to codon optimization for use in P. ohmeri ATCC 20209according to Table 7 of example 2, using the Optimizer program.

At the 5′ and 3′ ends of the resulting sequence, nucleotides encodingfor the recognition sites of the restriction enzymes AscI (GGCGCGCC) andSphI (GCATGC) respectively, were added in order to facilitate furthercloning.

Additionally, an adenosine triplet was included in front of the startATG to account for an adenosine at the −3 position in the Kozak-likesequence of yeasts.

The final sequence (SEQ ID NO: 1) was then submitted for synthesis(GeneArt, Regensburg, Germany).

The synthesized DNA fragment encoding the NAD⁺-specific D-arabitol4-oxidoreductase from E. coli was delivered as 5 μg lyophilized plasmidDNA in a pMK-RQ derived vector (12ABYWMP, FIG. 1).

For further sub-cloning the gene was released by restriction cuttingwith AscI and SphI enzymes (New England Biolabs, Ipswich, Mass.).

Example 4. Mutagenesis and Cloning of the Pichia stipitis NADH andNADPH-Specific Xylitol Dehydrogenase

Cloning of the Pichia stipitis NADH-Specific Xylitol Dehydrogenase Gene

The known nucleotide sequence of the yeast (Pichia stipitis) gene XYL2,encoding xylitol dehydrogenase (Kotter et al., Curr. Genet. 18:493-500(1990)) was cloned in the plasmidic vector lig 7.78 following theteaching of FR 2 765 589 (see example 4 and FIG. 7 of this patent). Therestriction map of the vector is presented in FIG. 2A.

Mutagenesis and Cloning of the Pichia stipitis NADPH-Specific XylitolDehydrogenase Gene

A DNA fragment encoding the NADPH-specific xylitol dehydrogenase XYL2from Pichia stipitis was chemically synthesized (GeneArt® GeneSynthesis, Life Technologies, Regensburg, Germany) according to thesequence of SEQ ID NO: 4.

Nucleotides 319 to 1410 of sequence X55392.1 (obtained from the NCBIGenBank database) coding for the XYL2 gene were used as template.

According to the paper from Watanabe et al. (J. Biol. Chem., 2005, 280,10340-10345), the cofactor preference of the xylitol dehydrogenase couldbe changed from NADH to NADPH by introducing four published amino acidmutations: D207A/I208R/F209S/N211R (numbering based on P22144 proteinsequence obtained from the UniProt database).

Accordingly, the codons encoding for D207, I208, F209 and N211 weremanually replaced by GCT, AGA, TCA and AGA in the correspondingsequence, respectively.

Additionally, nucleotides coding for the recognition sites of therestriction enzymes Hindlll (AAGCTT) and SacII (CCGCGG) were manuallyincluded at the respective 5′ and 3′ ends, in order to facilitatefurther cloning.

Furthermore, an adenosine triplet was included in front of the start ATGto account for an adenosine at the—3 position in the Kozak-like sequenceof yeasts. The final sequence (SEQ ID NO: 4) was submitted for synthesis(GeneArt, Regensburg, Germany).

The synthesized DNA fragment encoding the NADPH-specific xylitoldehydrogenase from P. stipitis was delivered as 5 μg lyophilized plasmidDNA in a pMA-T derived vector (12AALQTP, FIG. 2B).

Example 5. Mutagensis and Cloning, of the Gluconobacter oxydansNADPH-Specific Xylitol Dehydrogenase Gene

A DNA fragment encoding the NADPH-specific xylitol dehydrogenase Xdhfrom Gluconobacter oxydans was chemically synthesized (GeneArt® GeneSynthesis, Life Technologies, Regensburg, Germany), according to thesubmitted sequence of SEQ ID NO: 7.

Nucleotides 1063 to 1851 of sequence AB091690.1 (obtained from the NCBIGenBank database) coding for the Xdh gene were used as template andsubjected to codon optimization for use in P. ohmeri ATCC 20209according to Table 7 (Example 2) using the Optimizer program.

Based on the publication by Ehrensberger et al. (Structure, 2006, 14,567-575), the cofactor specificity of the enzyme could be changed fromNADH to NADPH by introducing two published amino acid mutations:D38S/M39R (numbering based on Q8GR61 protein sequence obtained from theUniProt database).

Thus, the codons encoding for D38 and M39 were manually replaced by TCTand AGA in the corresponding sequence, respectively. Additionally,nucleotides encoding for the recognition sites of the restrictionenzymes AscI (GGCGCGCC) and SphI (GCATGC) were manually included at therespective 5′ and 3′ ends, in order to enable further cloning.

Furthermore, an adenosine triplet was included in front of the start ATGto account for an adenosine at the −3 position in the Kozak-likesequence of yeasts. The final sequence (SEQ ID NO: 7), was submitted forsynthesis (GeneArt, Regensburg, Germany).

The synthesized DNA fragment encoding the NADPH-specific xylitoldehydrogenase from Gluconobacter oxydans was delivered as 5 μglyophilized plasmid DNA in a pMA-T derived vector (13AAYSYP, FIG. 3).For further subcloning, the gene was released by restriction cuttingwith AscI and SphI enzymes (New England Biolabs, Ipswich, Mass.).

Example 6. Construction of a P. ohmeri Vector for Heterologous GeneExpression Using the poURA3 Selection Marker

The Cloning of a Vector with Replaceable:

-   -   promoter,    -   open reading frame, and    -   terminator elements

was performed by two successive overlap PCRs of three individualfragments (FIG. 4).

The vector was originally planned as an expression model, to test thecloning and the overexpression of the tagatose 3-epimerase gene in therecombinant Pichia ohmeri strain.

As it will be described below, the tagatose 3-epimerase gene has beencloned into specific AscI-SphI restriction sites cassette, allowing thecloning of any gene of interest by using these same sites of insertion.

The cloning was conceived by the following way.

In a first PCR (PCR1), a 490 bp long ribulose reductase promoterfragment of P. ohmeri flanked by SpeI and AscI sites (underlined inprimer sequence) was amplified using:

primer EV2960: (SEQ ID No 12) GAACTAGTGGATCCGTAGAAATCTTG and primerEV2961: (SEQ ID No 13) CTTTGTTCATTTTGGCGCGCCTTTTAGTTTAATAAGGGTCCGTG

Additionally, at the 5′ end of the reverse primer EV2961, a 13nucleotide long fragment representing the 5′ end of thetagatose-3-epimerase gene was added. This fragment together with the 8nucleotides of the AscI site and the 10 following nucleotides of 3′ endof the ribulose reductase promoter were needed as overlap for fusing thefragment of PCR1 with the fragment of PCR2 described below. Genomic DNAof P. ohmeri ATCC 20209 was used as template.

For this purpose, a freshly streaked out P. ohmeri colony wasresuspended in 30 μl of 0.2% SDS and heated for 4 min at 95° C. Afterfull speed centrifugation, 0.5 μl of the supernatant was used for PCR.

The template was amplified in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 25 cycles with 10 sec at 98° C./20 sec at 50° C./15 secat 72° C., and a final extension step of 10 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.).

In a second PCR (PCR2), a 911 bp long fragment of thetagatose-3-epimerase of Pseudomonas cichorii ST24 flanked by AscI andSphI sites (underlined in primer sequence) was amplified using:

primer EV2962: (SEQ ID No 14) AAACTAAAAGGCGCGCCAAAATGAACAAAGTTGGCATG andprimer EV2963: (SEQ ID No 15) TTCTCTTCGAGAGCATGCTCAGGCCAGCTTGTCACG.

The 5′ end of primer EV2962 contains a 9 nucleotide long fragmentrepresenting the 3′ of the ribulose reductase promoter.

This fragment together with the 8 nucleotides of the AscI site and thefollowing 12 nucleotides of the tagatose-3-epimerase open reading frame,is used for the overlap PCR to fuse the PCR2 product to the previouslydescribed PCR1 product.

Additionally, the 5′ end of reverse primer EV2963 contains a 12nucleotide long fragment representing the 5′ end of the ribulosereductase terminator of P. ohmeri.

This fragment, together with the 6 nucleotides of the SphI site and thefollowing 12 nucleotides of the 3′ end of the tagatose-3-epimerase openreading frame, is needed as overlap for fusing PCR2 with the PCRfragment of PCR3 described below.

As template 25 ng of vector 12AAMCJP (FIG. 5) (GeneArt, Regensburg,Germany) containing a synthesized copy of the tagatose-3-epimarease geneof Pseudomonas cichorii ST24 was used (nucleotide 719 to 1591 ofAB000361.1, from the NCBI GenBank database) SEQ ID No: 11.

The template was amplified in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 25 cycles with 10 sec at 98° C./20 sec at 48° C./30 secat 72° C., and a final extension step of 10 minutes at 72° C.

In a third PCR (PCR3), a 380 bp long fragment of the ribulose reductaseterminator of P. ohmeri flanked by SphI and SacII sites (underlined inprimer sequence) was amplified using:

primer EV2964 (SEQ ID No 16) AAGCTGGCCTGAGCATGCTCTCGAAGAGAATCTAG andprimer EV2965 (SEQ ID No 17) GTTCCGCGGAGAATGACACGGCCGAC

The 5′ end of primer EV2964 contains a 12 nucleotide long fragment ofthe 3′ end of the tagatose-3-epimerase open reading frame that, togetherwith the 6 nucleotides of the SphI site and the following 12 nucleotidesof the ribulose reductase terminator of P. ohmeri is used for the fusionof PCR3 to the previously described PCR2.

Genomic DNA of P. ohmeri ATCC 20209 was used as template. After fullspeed centrifugation, 0.5 μl of the supernatant was used in PCR. Forthis purpose, a freshly streaked out P. ohmeri colony was resuspended in30 μl of 0.2% SDS and heated for 4 min at 95° C.

The template was amplified in a reaction mix consisting of 200 μM ofeach dNTP, 0.5 μM of each primer and 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 25 cycles with 10 sec at 98° C./20 sec at 50° C./15 secat 72° C., and a final extension step of 10 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.). The PCR product was separated on a 1% agarose gel, extractedand purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.).

Fusion of the three individual PCR fragments was performed as follows:50 ng of each gel purified product of PCR1 and PCR2 was used as templatein a PCR reaction with EV2960 and EV2963.

A 30 nucleotide long homologous segment in the two fragments, resultingfrom the primer design described above, was used as overlap in thefusion reaction.

This way, a 1.4 kb long fragment, consisting of a ribulose reductasepromoter of P. ohmeri flanked by SpeI and AscI sites was fused to theopen reading frame of the tagatose-3-epimerase of Pseudomonas cichoriiST24.

The templates were amplified in a reaction mix consisting of 200 μM ofeach dNTP, 0.5 μM of each primer and 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C., followed by 30 cycles with 10 sec at 98° C./20 sec at 62° C./45 secat 72° C., and a final extension step of 10 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.).

The purified fragment was fused in a second overlap PCR to the productof PCR3. 40 ng of each fragment was used as template and amplified withEV2960 and EV2965.

A 30 nucleotide long homologous segment in the two fragments, resultingfrom the primer design described above, was used as overlap in thefusion.

This way, a 1.8 kb long fragment, consisting of a ribulose reductasepromoter of P. ohmeri flanked by SpeI and AscI and the open readingframe of the tagatose-3-epimerase of Pseudomonas cichorii ST24 flankedby AscI and SphI sites was fused to the ribulose reductase terminator ofP. ohmeri.

The templates were amplified in a reaction mix consisting of 200 μM ofeach dNTP, 0.5 μM of each primer and 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 30 cycles with 10 sec at 98° C./20 sec at 65° C./55 secat 72° C., and a final extension step of 10 minutes at 72° C. The PCRproduct was separated on an agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.).

The final PCR product consisting of a 1.7 kb long fragment of thetagatose-3-epimerase of Pseudomonas cichorii ST24 flanked by a ribulosereductase promoter and terminator was digested with restriction enzymesSpeI and Seen (New England Biolabs, Ipswich, Mass.), gel purified andligated overnight at 16° C. with a 9.8 kb long isolated SpeI/SacIIfragment of a lig7.78 vector backbone using T4 DNA ligase (New EnglandBiolabs. Ipswich, Mass.) (FIG. 6).

After transformation of XL.10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.). The purified plasmid DNA was used forfurther characterization by restriction digestion and sequencing(Microsynth, Balgach, Switzerland).

The newly cloned expression plasmid pEVE2523 (FIG. 7) is a shuttle E.coli-P. ohmeri vector consisting of a bacterial (E. coli) origin ofreplication and an ampicillin resistance gene, the yeast (P. ohmeri)autonomous replication sequence, and the poURA3 (P. ohmeri) gene forselection in yeast.

Moreover, it contains an exchangeable P. ohmeri ribulose reductasepromoter element (via SpeI and AscI restriction) and terminator element(via SphI and SacII) flanking an open reading frame of thetagatose-3-epimerase of Pseudomonas cichorii (exchangeable via AscI andSphI restriction).

Example 7. Construction of a P. ohmeri Vector for Heterologous GeneExpression Using the poLEU2 Selection Marker

For the construction of a second P. ohmeri expression vector, theexpression cassette of plasmid pEVE2523 (FIG. 7) described previously inExample 6 was cloned into a vector containing the P. ohmeri poLEU2selection marker (FIG. 6).

A blunted 1.7 kb fragment of vector pEVE2523 (FIG. 7) cut with SpeI andSacII (New England Biolabs, Ipswich, Mass.) was used as insert. Bluntingwas performed with the Blunting Enzyme Mix (New England Biolabs,Ipswich, Mass.) for 15 min at room temperature, followed by heatinactivation of the enzymes for 10 min at 70° C.

The vector backbone was obtained from a poARS vector (plig3-FR 2772788)linearized with SalI (New England Biolabs, Ipswich, Mass.), blunted anddephosphorylated for 1 h at 37° C. using Antarctic phosphatase (NewEngland Biolabs, Ipswich, Mass.). Gel purified insert and vectorbackbone using Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.) were ligated for 1 h at RT using T4 DNAligase (New England Biolabs, Ipswich, Mass.).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and used for further characterization byrestriction digestion and sequencing (Microsynth, Balgach, Switzerland).

The new cloned expression plasmids pEVE2560 (FIG. 8) is a shuttle E.coli-P. ohmeri vector containing a bacterial (E. coli) origin ofreplication and an ampicillin resistance gene, the yeast (P. ohmeri)autonomous replication sequence, and the poLEU2 (P. ohmeri) gene forselection in yeast.

Moreover, the open reading frame of the tagatose-3-epimerase ofPseudomonas cichorii flanked by a P. ohmeri ribulose reductase promoterand terminator is exchangeable via AscI and SphI restriction.

Example 8. Construction of a P. ohmeri Vector for Overexpression ofGluconobacter oxydans NADPH-Specific Xylitol Dehydrogenase

A P. ohmeri vector for overexpression of Gluconobacter oxydansNADPH-specific xylitol dehydrogenase was constructed.

For cloning into the expression vector, the DNA fragment encoding theGluconobacter oxydans NADPH-specific xylitol dehydrogenase was releasedfrom vector 13AAYSYP (FIG. 3) by cutting with AscI and SphI restrictionenzymes (New England Biolabs, Ipswich, Mass.).

The 803 bp fragment was gel-purified using Zymoclean™ Gel DNA RecoveryKit (Zymo Research Corporation, Irvine, Calif.) and ligated for 2 h atroom temperature to the 9.8 kb AscI/SphI-digested and gel-purifiedvector backbone of pEVE2523 (FIG. 7) using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.) (FIG. 9).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3284 (FIG. 10) contains the codon-optimizedNADPH-specific xylitol dehydrogenase of Gluconobacter oxydans flanked bya ribulose reductase promoter and terminator of P. ohmeri and the poURA3selection marker.

Example 9. Construction of a P. ohmeri Vector for Overexpression ofPichia stipitis NADPH-Specific Xylitol Dehydrogenase

For sub-cloning into the expression vector, the DNA fragment encodingthe NADPH-specific xylitol dehydrogenase from Pichia stipitis had to beflanked with AscI and SphI restriction sites.

For this purpose:

EV3101 primer (SEQ ID No 18)AAGGCGCGCCAAA ATGACTGCTAACCCTTCC containing an AscI site (underlined)and EV3102 primer (SEQ ID No 19)GAGCATGCTTACTCAGGGCCGTCAATG containing a SphI (underlined)

were used in a PCR reaction with 30 ng of vector 12AALQTP (FIG. 2B) astemplate.

The template was amplified in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 25 cycles with 10 sec at 98° C./20 sec at 55° C./30 secat 72° C., and a final extension step of 10 minutes at 72° C.

The 1.1 kb PCR product was separated on a 1% agarose gel, extracted,purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.) and restriction digested with AscI and SphI(New England Biolabs, Ipswich, Mass.). After column purification withthe DNA Clean & Concentrator™-5 Kit (Zymo Research Corporation, Irvine,Calif.), it was ligated for 2 h at room temperature to the 10.6 kbAscI/SphI-digested and gel-purified vector backbone of pEVE2523 (FIG. 7)and the 11.8 kb AscI/SphI-digested and gel-purified vector backbone ofpEVE2560 (FIG. 8) respectively, using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.) (FIG. 11).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated and further characterized by restriction digestion andsequencing (Microsynth, Balgach, Switzerland).

The resulting plasmids pEVE2562 and pEVE2564 (FIG. 12) contain the codonoptimised NADPH-specific xylitol dehydrogenase of Pichia stipitisflanked by a ribulose reductase promoter and terminator of P. ohmeri andeither the poURA3 or poLEU2 selection marker, respectively.

Example 10. Construction of a P. ohmeri Vector for Overexpression ofPichia stipitis NADH-Specific Xylitol Dehydrogenase

For sub-cloning into the expression vector, the DNA fragment encodingthe NADH-specific xylitol dehydrogenase from Pichia stipitis had to beflanked with AscI and SphI restriction sites.

For this purpose:

EV3101 (SEQ ID No 18) (AAGGCGCGCCAAA ATGACTGCTAACCCTTCC)containing an AscI site (underlined) and EV3102 (SEQ ID No 19)(GA GCATGCTTACTCAGGGCCGTCAATG) containing a SphI (underlined)

were used in a PCR reaction with 30 ng of vector lig7.78 (FIG. 2A) astemplate.

The template was amplified in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was performed with an initial denaturation step of 30 sec at 98°C. followed by 25 cycles with 10 sec at 98° C./20 sec at 55° C./30 secat 72° C., and a final extension step of 10 minutes at 72° C.

The 1.1 kb PCR product was separated on a 1% agarose gel, extracted,purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.) and restriction digested with AscI and SphI(New England Biolabs, Ipswich, Mass.).

After column purification with the DNA Clean & Concentrator™-5 Kit (ZymoResearch Corporation, Irvine, Calif.), it was ligated for 2 h at roomtemperature to the 10.5 kb AscI/SphI-digested and gel-purified vectorbackbone of pEVE2560 (FIG. 8) using T4 DNA ligase (New England Biolabs,Ipswich, Mass.) (FIG. 13).

After transformation of XL10 Gold uitracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated and further characterized by restriction digestion andsequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2563 (FIG. 14) contains the codon optimisedNADH-specific xylitol dehydrogenase of Pichia stipitis flanked by aribulose reductase promoter and terminator of P. ohmeri and the poLEU2selection marker.

Example 11. Construction of P. ohmeri Vectors for Overexpression of E.coli NAD⁺-Specific D-Arabitol 4-Oxidoreductase

A P. ohmeri vector for overexpression of E. coli NAD⁺-specificD-arabitol 4-oxidoreductase was constructed.

For cloning into the expression vector, the DNA fragment encoding thecodon-optimised E. coli NAD⁺-specific D-arabitol 4-oxidoreductase wasreleased from vector 12ABYWMP (FIG. 1) by cutting with AscI and SphIrestriction enzymes (New England Biolabs, Ipswich, Mass.).

The 1.4 kb fragment was gel-purified using Zymoclean™ Gel DNA RecoveryKit (Zymo Research Corporation, Irvine, Calif.) and ligated for 2 h atroom temperature to the 9.8 kb AscI/SphI-digested and gel-purifiedvector backbone of pEVE2523 (FIG. 7) using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.) (FIG. 15).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2839 (FIG. 16) contains the codon-optimised E.coli NAD⁺-specific D-arabitol 4-oxidoreductase flanked by a ribulosereductase promoter and terminator of P. ohmeri and the poURA3 selectionmarker.

In addition to the P. ohmeri ribulose reductase promoter, theNAD⁺-specific D-arabitol 4-oxidoreductase from E. coli was also clonedunder the control of the P. ohmeri phosphoglycerate kinase (poPGK1)promoter and transketolase (poTKL) terminator.

Cloning was performed in two consecutive steps, by first replacing theribulose reductase promoter by the poPGK1 promoter, followed by anexchange of the ribulose reductase terminator for the poTKL terminator.

A 611 bp long fragment of the P. ohmeri poPGK1 promoter was amplifiedfrom genomic DNA of P. ohmeri using:

primer EV3177 (SEQ ID No 20) (GAAGACTAGTTCACGTGATCTC) containing aSpeI site (underlined) and primer EV3178 (SEQ ID No 21)(CACT GGCGCGCCTTTTGTGTGGTGGTGTCC), containing an AscI site (underlined).

The genomic DNA template was prepared by resuspending a freshly streakedout P. ohmeri colony in 30 μl of 0.2% SDS and heating for 4 min at 95°C. After full speed centrifugation, 0.5 μl of the supernatant was usedfor PCR.

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was accomplished with an initial denaturation step of 2 min at96° C. followed by 25 cycles with 10 sec at 96° C./10 sec at 58° C./30sec at 72° C., and a final extension step of 2 minutes at 72° C.

The PCR product was separated on a 1% agarose gel, extracted andpurified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.).

The amplified 610 bp long poPGK1 promoter fragment was restrictiondigested with SpeI and AscI (New England Biolabs, Ipswich, Mass.) andligated for 2 h at room temperature to the 11.5 kb SpeI/AscI-digestedand gel-purified vector backbone of pEVE2839 (FIG. 16) using T4 DNAligase (New England Biolabs, Ipswich, Mass.) (FIG. 17).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3102 (FIG. 18) contains the codon-optimised E.coli NAD⁺-specific D-arabitol 4-oxidoreductase flanked by aphosphoglycerate kinase (poPGK1) promoter and ribulose reductaseterminator of P. ohmeri and the poURA3 selection marker.

In the next step the ribulose reductase terminator of pEVE3102 wasexchanged for the tranketolase (poTKL) terminator of P. ohmeri.

A 213 bp long fragment of the P. ohmeri poTKL terminator was amplifiedfrom genomic DNA of P. ohmeri using:

primer EV3817 (SEQ ID No 22) (TAGCAGCATGCATAGGTTAGTGAATGAGGTATG)containing a SphI site (underlined) and (SEQ ID No 23)primer EV3818 (TAGGTCCGCGGGAGCTTCGTTAAAGGGC)containing a SacII site (underlined).

The genomic DNA template was prepared as described above.

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer.

The PCR was accomplished with an initial denaturation step of 2 min at96° C. followed by 25 cycles with 10 sec at 96° C./10 sec at 57° C./30sec at 72° C., and a final extension step of 2 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.).

The amplified 213 bp long poTKL terminator fragment was restrictiondigested with SphI and SacII (New England Biolabs, Ipswich, Mass.) andligated for 2 h at room temperature to the 11.5 kb SphI/SacII-digestedand gel-purified vector backbone of pEVE3102 (FIG. 18) using T4 DNAligase (New England Biolabs, Ipswich, Mass.) (FIG. 17).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3123 (FIG. 19) contains the codon-optimised E.coli NAD⁺-specific D-arabitol 4-oxidoreductase flanked by aphosphoglycerate kinase (poPGK1) promoter and a transketolase (poTKL)terminator of P. ohmeri and the poURA3 selection marker.

In order to be able to express the NAD⁺-specific D-arabitol4-oxidoreductase of E. coli from a plasmid using another selection, thepoURA3 marker of pEVE3123 was exchanged for the poLEU2 marker.

For this purpose the poURA3 marker was released from vector pEVE3123(FIG. 19) by restriction digestion with PsiI and AfeI (New EnglandBiolabs, Ipswich, Mass.).

The 9.1 kb vector backbone was gel-purified using Zymoclean™ Gel DNARecovery Kit (Zymo Research Corporation, Irvine, Calif.), blunted withthe Blunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.) for 15min at room temperature, followed by heat inactivation of the enzymesfor 10 min at 70° C. and dephosphorylated for 1 h at 37° C. usingAntarctic phosphatase (New England Biolabs, Ipswich, Mass.).

As insert, a 3 kb blunted and gel-purified fragment of the poLEU2 markerreleased from vector pEVE2560 (FIG. 8) by AseI and AfeI restrictiondigestion was used, Ligation of the fragments was performed for 2 h atroom temperature using T4 DNA ligase (New England Biolabs, Ipswich,Mass.) (FIG. 20).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3157 (FIG. 21) contains the codon-optimised E.coli NAD-specific D-arabitol 4-oxidoreductase flanked by aphosphoglycerate kinase (poPGK1) promoter and a transketolase (poTKL)terminator of P. ohmeri and the poLEU2 selection marker.

Example 12. Expression of the Plasmidic E. coli NAD⁺-Specific D-arabitol4-Oxidoreductase and of the Plasmidic Pichia stipitis NADPH-SpecificXylitol Dehydrogenase Gene in Pichia ohmeri Strain ATCC 20209

For the biosynthetic conversion of arabitol into xylitol, thesimultaneous expression of the NAD⁺-specific E. coli D-arabitol4-oxidoreductase and the NADP-specific xylitol dehydrogenase of P.stipitis is necessary.

The first enzyme leads to the formation of xylulose and the second onesconvert xylulose into xylitol.

P. ohmeri strain SRLU (MATh⁻ leu2 ura3) derived from ATCC 20209 andauxotrophic for leucine and uracil (Piredda and Gaillardin, 1994, supra)was used as host for the construction of a yeast strains secretingxylitol by transformation with plasmids:

-   -   pEVE2839 (NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli)        and    -   pEVE2564 (NADPH-specific xylitol dehydrogenase of P. stipitis)

leading to strain EYS2755

Additionally, as a control (following the teaching of WO 94/10325) astrain expressing the NADH-specific wild type xylitol dehydrogenase ofP. stipitis was also constructed by transformation with plasmids:

-   -   pEVE2839 (NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli)        and    -   pEVE2563 (NADH-specific xylitol dehydrogenase of P. stipitis)

into the SRLU host, leading to strain EYS2962.

As control, strains transformed with the single plasmids:

-   -   pEVE2839 (NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli),    -   pEVE2563 (NADH-specific xylitol dehydrogenase of P. stipitis),        and    -   pEVE2564 (NADPH-specific xylitol dehydrogenase of P. stipitis)

leading to EYS2943, EYS2696 and EYS2697 respectively, were alsogenerated.

Yeast transformation was carried out in essential by the spheroplastingmethod of Green et al. (Green E D., Hieter, P., and Spencer F. A.,chapter 5 in Genome Analysis: A Laboratory Manual, Vol. 3, CloningSystems, Birren et al. (eds.), Cold Spring Harbor Press, New York, 1999)with the following modifications: Instead of Lyticase, Zymolyase 100Twas used for generation of spheroplasts and the incubation with theenzyme was performed at 37° C. until the OD of the cell suspensionreached 20-30% of the original OD before Zymolyase treatment.

Briefly, P. ohmeri cells were grown overnight at 30° C. in YPD medium(Yeast extract 1% (w/v), Peptone 2% (w/v), Dextrose 2% (w/v)) to a finalOD₆₀₀ of 3-5.

200 OD₆₀₀ units were harvested by centrifugation, washed once with waterand 1M sorbitol, and resuspended in SCE buffer (1 M sorbitol, 100 mMcitric acid trisodium salt dihydrate, 10 mM EDTA) to a finalconcentration of 70 ODs/ml.

DTT and Zymolase (LuBio Science, Luzern, Switzerland) were added to afinal concentration of 10 mM and 0.5 U/OD, respectively and the mixtureincubated at 37° C. with slow shaking.

The cell wall digestion was followed by measuring the optical density ofthe solution diluted in water. When this value dropped to 80% of theoriginal, the digestion was terminated by careful centrifugation andwashing with 1 M sorbitol and STC buffer (0.98 M sorbitol, 10 mM Tris pH7.5, 10 mM CaCl₂).

Speroplasts were carefully resuspended in STC buffer containing 50 μg/mlcalf-thymus DNA (Calbiochem/VWR, Dietikon, Switzerland) to a finalconcentration of 200 OD/ml. Aliquots of 100 μl were mixed with 100-200ng of plasmid DNA and incubated for 10 min at room temperature.

1 ml PEG solution (19.6% PEG 8000 w/v, 10 mM Tris pH 7.5, 10 mM CaCl₂)was added to the suspension, incubated for 10 minutes and pelleted.Spheroplasts were regenerated at 30° C. for 1-2 h in 1 ml of a 1 Msorbitol solution containing 25% YPD and 7 mM CaCl₂.

To the regenerated cells 7 ml of 50° C. warm top agar (0.67% yeastnitrogen base w/o amino acids, 0.13% drop-out powder withoutleucine/uracil/histidine/tryptophan/methionine, 0.086%₀ of requiredmissing amino acid, 2% glucose, 1 M sorbitol, pH5.8 and 2.5% Noble agar)was added and the mixture was poured evenly onto pre-warmed, sorbitolcontaining selective plates (0.67% yeast nitrogen base w/o amino acids,0.13% drop-out powder withoutleucine/uracil/histidine/tryptophan/methionine, 0.086% of requiredmissing amino acid, 2% glucose, 1 M sorbitol, pH5.8).

Plates were incubated for 3-5 days at 30° C. Transformants werereselected on the appropriate selective plates.

Each generated strain was tested in triplicates for arabitol, xylitoland ribitol production.

For this purpose clones were first grown at 30° C. overnight in seedmedia (0.67% yeast nitrogen base without amino acids; 0.13% drop-outpowder without leucine/uracil/histidine/tryptophan/methionine; 0.086%₀of required missing amino acid; 5% glucose; pH5.7).

Out of this overnight culture a main culture in production media (0.67%yeast nitrogen base without amino acids; 0.13% drop-out powder withoutleucine/uracil/histidine/tryptophan/methionine; 0.086%₀ of requiredmissing amino acid; 15% glucose; pH5.7) at a starting OD600 of 0.2 wasinoculated.

This culture was grown at 37° C. for 48 hours and the arabitol, xylitoland ribitol concentrations of the supernatants were determined byHPLC/MS using a Aminex® HPX-87 column (Bio-Rad, Hercules, Calif.) and aWaters® TQ-Detector (Acquity® UPLC linked to a triple quadrupoldetector, Waters, Milford, Mass.) and isocratic conditions with 100%water as mobile phase.

Polyol titers of all tested strains are depicted in Table 8.

TABLE 8 Polyol production of P. ohmeri SRLU strains transformed withNADH- and NADPH-specific xylitol dehydrogenase of P. stipitis and/orwith NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli (average oftriplicates) Strain Arabitol (g/L) Xylitol (g/L) Ribitol (g/L) SRLU 32.9± 2.4 nd nd EYS2943 [pEVE2839] 26.4 ± 2.8 2.3 ± 0.1 0.7 ± 1.2 EYS2696[pEVE2563] 36.0 ± 2.7 nd. 0.8 ± 0.1 EYS2697 [pEVE2564] 31.1 ± 1.6 nd 6.3± 0.1 EYS2962 [pEVE2839/ 29.6 ± 0.8 7.0 ± 0.3 2.3 ± 0.1 pEVE2563]EYS2755 [pEVE2839/ 16.4 ± 2.2 19.9 ± 0.8  10.9 ± 0.4  pEVE2564] nd—notdetected

Use of the NADPH-specific xylitol dehydrogenase of P. stipitis leads toa significant increase in xylitol titers, as compared to the wild typeNADH-specific enzyme.

Example 13. Expression of the Plasmidic Gluconobacter oxydansNADPH-Specific Xylitol Dehydrogenase Gene in Pichia ohmeri

In addition to a xylitol producing strain using the NADP-specificxylitol dehydrogenase of P. stipitis a second strain expressing theNADP-specific xylitol dehydrogenase of G. oxydans was engineered.

P. ohmeri strain SRLU (MATh⁻ leu2 ura3) derived from ATCC 20209 andauxotrophic for leucine and uracil (Piredda and Gaillardin, 1994, supra)was used as host for the construction of a yeast strains secretingxylitol by transformation with plasmids pEVE3157 (NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli) and pEVE3284 (NADPH-specificxylitol dehydrogenase of G. oxydans) leading to strain EYS3324.

As control, strains transformed with the single plasmids:

-   -   pEVE3157 (NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli)        and    -   pEVE3284 (NADH-specific xylitol dehydrogenase of G. oxydans),        leading to EYS3067 and EYS3323 respectively, were also        generated.

The E. coli D-arabitol 4-oxidoreductase used for the construction of theabove strains is controlled by poPGK1 promoter in contrast to the poRRpromoter used in strains expressing the xylitol dehydrogenase of P.stipitis.

However, to exclude a promoter influence and therefore, to be able tocompare polyol levels in strains expressing the xylitol dehydrogenasefrom G. oxydans with those expressing the corresponding enzyme from P.stipitis, an additional strain has been generated.

This strain EYS2963 was obtained by transforming the SRLU host with

-   -   pEVE3123 (NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli)        and    -   pEVE2564 (NADPH-specific xylitol dehydrogenase of P. stipitis).

Yeast transformation was carried out as described in Example 12. Eachgenerated strain was tested in triplicates for arabitol, xylitol andribitol production as described in Example 12.

Polyol titers of all tested strains are depicted in Table 9.

TABLE 9 Polyol production of P. ohmeri SRLU strains transformed withNADPH-specific xylitol dehydrogenase of G. oxydans and/or withNAD⁺-specific D-arabitol 4-oxidoreductase of E. coli (average oftriplicates) Strain Arabitol (g/L) Xylitol (g/L) Ribitol (g/L) SRLU 32.9± 2.4 nd nd EYS3067 [pEVE3157] 29.0 ± 3.8  1.5 ± 0.3 1.8 ± 0.4 EYS3323[pEVE3284] 32.8 ± 0.6 nd nd EYS3324 [pEVE3157/ 26.3 ± 1.7 21.1 ± 1.1 1.2± 0.1 pEVE3284] EYS2963 [pEVE3123/ 27.3 ± 2.5 17.7 ± 1.7 13.9 ± 0.7 pEVE2564] nd—not detected

Xylitol titers in strains expressing the NADPH-specific xylitoldehydrogenase from G. oxydans (EYS3324) are similar to those of strainsexpressing the corresponding enzyme from P. stipitis (EYS2963). However,the G. oxydans enzyme leads to much lower ribitol titers, thus showing ahigher substrate specificity towards xylulose.

Example 14. Generation of a Mutant P. ohmeri Strain with IncreasedArabitol Secretion

A higher arabitol producer mutant has been selected from an UVirradiated suspension of P. ohmeri ATCC 20209.

The UV-irradiation system (Vilber Lourmat, France), was equipped with amicroprocessor-controlled RMX-3 W radiometer. P. ohmeri was grown on YPDagar (Dextrose 20 g/L) at 37° C. overnight.

A suspension was prepared to reach 10⁶ cfu/mL (OD₆₂₀=0.4) and 5 mL wereput into a sterile Petri dish. The suspension was irradiated afterremoving the cover from the dish. The UV wavelength was 254 nm and theirradiation energy was 1.8 10⁻² J/cm². 90% of mortality of the yeastcells was obtained. After stopping the irradiation and replacing the lidon the dish, the suspension was transferred into a sterile tube locatedinto an iced bath.

20 mL of YPD liquid medium was inoculated with the mutated suspensionand was incubated for 12 hours at 37° C., 250 rpm.

After incubation the mutated culture was diluted with sterile 40%glycerol (V/V). Aliquots were distributed into 5 mL vials and frozen at−80° C.

The screening was based on the osmophilic property of Pichia ohmeriwhich is able to grow on very high concentrations of Dextrose (up to 600g/L).

Our goal was to select mutants able to grow faster than the motherstrain on YPD agar containing Dextrose 600 g/L or 700 g/L.

Defrosted aliquots were spread on YPD₆₀₀ and YPD₇₀₀ and the firstappearing colonies were selected and tested for the production ofarabitol in shake flasks.

The subculture and production medium were made of glucose 50 g/L or 100g/L respectively, yeast extract 3 g/L, MgSO₄ 1 g/L and KH₂PO₄ 2 g/L, pH5.7. The subculture (10 mL in a 100 mL flask) was incubated for 24 h at37° C., 250 rpm. The production (40 mL in a 500 mL flask) was inoculatedby 5 mL of subculture and incubated for 64 hours at 37° C., 250 rpm.

Glucose g/L 64 h Arabitol g/L 64 h P. ohmeri ATCC 20209 6.0 52.7 P.ohmeri CNCM I-4605 0 58.6

The mutant P. ohmeri strain was selected for its faster consumption ofglucose and its higher production of arabitol and was deposited inFrance on Mar. 7, 2012, with the Collection Nationale de Cultures deMicroorganismes [National Collection of Microorganism Cultures] of theInstitut Pasteur (CNCM), 25 rue du Docteur Roux, 75724 PARIS Cedex 15,under number 1-4605.

Example 15. Construction of a LEU2 Deletion Plasmid

In order to be able to use the newly generated CNCM I-4605 strain forplasmid selection and gene integrations, a plasmid for the deletion ofthe LEU2 open reading frame was constructed.

In a first step, a general integration vector that can be used in P.ohmeri was adapted from the S. cerevisiae CRE/loxP system. The vectorbackbone was isolated from pUG73 (Gueldener et al., 2002, Nucleic AcidRes, 30, e23) by restriction cutting with PstI and EcoRV enzymes (NewEngland Biolabs, Ipswich, Mass.).

As insert served a PCR fragment containing a LEU2 selection marker of P.ohmeri flanked by loxP sites, generated with primer pair:

EV3043 (SEQ ID No 24) (CACTGGCGCGCCCACTGCATGCGTCGACAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTAGACACATCGTGGATCCAAG CTATCAACGAGAGAGTC)and EV3044 (SEQ ID No 25) (AGTGGCTAGCAGTGCCATGGCCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTAAGGGTTCTCGAGACGCGTCATCTAGCATCTCATCTAC CAACTC) andpoARS (plig3 - FR 2772788 - see FIG. 6) as template.

The forward primer EV3043 contains an AscI (underlined) site preceding aSphI site (underlined), followed by a 48 bp long loxP fragment (bold)and a DraIII site (underlined). The 3′ end of EV3043 contains anadditional a 25 bp long fragment for amplification of the P. ohmeri LEU2gene. The reversed primer EV3044 on the other hand, contains a NheI(underlined) site preceding a NcoI site (underlined), followed by a 48bp long loxP fragment (bold) and a MluI site (underlined). The 3′ end ofEV3044 contains an additional a 25 bp long fragment for amplification ofthe P. ohmeri LEU2 gene. The template was amplified in a reaction mixconsisting of 200 μM of each dNTP and 0.5 μM of each primer with 0.02U/μl of iProof™ polymerase (BIO-RAD, Hercules, Calif.) in theappropriate 1× buffer. The PCR was performed with an initialdenaturation step of 30 sec at 98° C. followed by 30 cycles with 10 secat 98° C./10 sec at 65° C./50 sec at 72° C., and a final extension stepof 7 minutes at 72° C. The PCR product was separated on a 1% agarosegel, extracted and purified using the Zymoclean™ Gel DNA Recovery Kit(Zymo Research Corporation, Irvine, Calif.)

The amplified fragment was flanked by a PstI and EcoRV site in a secondPCR reaction for further subcloning. Amplification was performed with:

primer EV3056 (SEQ ID No 26) (CACTCTGCAGCACTGGCGCGCCCACTGCAT)containing the PstI site (underlined) and primer EV3057 (SEQ ID No 27)(CACTGATATCAGTGGCTAGCAGTGCCATGG) containing the EcoRV site (underlined)

in a reaction mix consisting of 200 μM of each dNTP and 0.5 μM of eachprimer with 0.02 U/μl of iProof™ polymerase (BIO-RAD, Hercules, Calif.)in the appropriate 1× buffer. The PCR was accomplished with an initialdenaturation step of 30 sec at 98° C. followed by 30 cycles with 10 secat 98° C./45 sec at 72° C., and a final extension step of 7 minutes at72° C. The PCR product was separated on a 1% agarose gel, extracted andpurified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.).

The amplified 2.5 kb LEU2 marker was restriction digested with PstI andEcoRV enzymes (New England Biolabs, Ipswich, Mass.), gel-purified withthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.) and ligated for 2 h at room temperature to the 2.4 kb PstI/EcoRV(New England Biolabs, Ipswich, Mass.), gel-purified (Zymoclean™ Gel DNARecovery Kit—Zymo Research Corporation, Irvine, Calif.) backbone ofvector pUG73 (Gueldener et al., 2002 Nucleic Acid Res, 30, e23) using T4DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG. 22). Aftertransformation of XL10 Gold ultracompetent cells (Agilent Technologies,Santa Clara, Calif.) with the ligation mixture, plasmid DNA was isolatedusing the Zyppy™ Plasmid Miniprep Kit (Zymo Research Corporation,Irvine, Calif.) and further characterized by restriction digestion andsequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2787 (FIG. 23) contains the P. ohmeri LEU2selection marker under the control of the endogenous promoter andterminator, flanked by two loxP sites. Additionally, a AscI and SphIsite have been introduced upstream of the first loxP and a NheI and NcoIsite downstream of the second loxP, in order to help in cloning ofregions homologous to the integration sites in the genome.

The LEU2 marker of the integration vector was then replaced by the nat1resistance gene of Streptomyces noursei, in a second cloning step, sincea deletion of the endogenous LEU2 open reading frame was aimed.

A DNA fragment encoding the nat1 gene of Streptomyces noursei waschemically synthesized by GeneArt® Gene Synthesis (Life Technologies,Regensburg, Germany) according to the submitted sequence of SEQ ID No28.

Nucleotides 204 to 776 of sequence S60706.1 (obtained from the NCBIGenBank database) coding for the nat1 gene were used as template andsubjected to codon optimization for use in P. ohmeri ATCC 20209according to Table 7 (above), using the Optimizer program.

At the 5′ and 3′ ends of the resulting sequence, nucleotides encodingfor the recognition sites of the restriction enzymes AscI (GGCGCGCC) andSphI (GCATGC) respectively, were manually added in the text file, inorder to facilitate further cloning. Additionally, an adenosine tripletwas included in front of the start ATG to account for an adenosine atthe −3 position in the Kozak-like sequence of yeasts.

The final sequence (SEQ ID No 28) was then submitted for synthesis toGeneArt (Regensburg, Germany). The synthesized DNA fragment encoding thenat1 gene was delivered as 5 μg lyophilized plasmid DNA in a pMA-Tderived vector (12ABTV4P, FIG. 24).

For the cloning of the net gene a vector containing a ribulose reductase(poRR) promoter and terminator was used. The terminator was exchanged byan orotidine-5′-phosphate decarboxylase (poURA3) terminator and the nat1gene was introduced between the promoter and terminator sequences.

For this purpose, the orotidine-5′-phosphate decarboxylase (poURA3)terminator was generated by PCR with:

primer EV3393 (SEQ ID No 29) (CAAGCATGCGGGAATGATAAGAGACTTTG)containing a SphI site (underlined) and primer EV3394 (SEQ ID No 30)(GGACCGCGGAAAGGTGAGGAAGTATATGAAC) containing a SacII site (underlined)and pEVE2523 (FIG. 7) as template.

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. The PCR wasaccomplished with an initial denaturation step of 30 sec at 98° C.followed by 30 cycles with 10 sec at 98° C./10 sec at 59° C./10 sec at72° C., and a final extension step of 5 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.). The 239 bp poURA3 terminator was restriction digested with SphIand SacII enzymes (New England Biolabs, Ipswich, Mass.) and ligated for2 h at room temperature to the 11 kb vector backbone of pEVE2681linearized with SphI and SacII restriction enzymes (New England Biolabs,Ipswich, Mass.) and gel-purified with Zymoclean™ Gel DNA Recovery Kit(Zymo Research Corporation, Irvine, Calif.) using T4 DNA ligase (NewEngland Biolabs, Ipswich, Mass.). After transformation of XL10 Goldultracompetent cells (Agilent Technologies, Santa Clara, Calif.) withthe ligation mixture, plasmid DNA was isolated using the Zyppy™ PlasmidMiniprep Kit (Zymo Research Corporation, Irvine, Calif.) and furthercharacterized by restriction digestion and sequencing (Microsynth,Balgach, Switzerland).

In a second cloning step, the nat1 gene was released from 12ABTV4P (FIG.24) by restriction cutting with SphI and AscI enzymes (New EnglandBiolabs, Ipswich, Mass.). Additionally, a blunting of the SphI site withthe Blunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.) for 15min at room temperature, followed by heat inactivation of the enzymesfor 10 min at 70° C. was performed in between the SphI and AscIdigestion. The 587 bp gel-purified fragment (Zymoclean™ Gel DNA RecoveryKit—Zymo Research Corporation, Irvine, Calif.) was than ligated to thegel-purified 10.5 kb vector backbone of the vector described above cutwith SphI and AscI restriction enzymes (New England Biolabs, Ipswich,Mass.).

Also the SphI site of the vector was blunted for 15 min at roomtemperature with the Blunting Enzyme Mix kit (New England Biolabs,Ipswich, Mass.), followed by a heat inactivation step of 10 min at 70°C. before the digestion with AscI was performed. Additionally, thevector was dephosphorylated for 1 h at 37° C. using Antarcticphosphatase (New England Biolabs, Ipswich, Mass.). The ligation wasperformed for 2 h at room temperature using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2798 (FIG. 25) contains the nat1 drugresistance marker flanked by a P. ohmeri ribulose reductase (poRR)promoter and an orotidine-5′-phosphate decarboxylase (poURA3)terminator.

The nat1 expression cassette was used to replace the P. ohmeri LEU2selection marker in the integrative vector. In order to facilitatefurther cloning the nat1 cassette had to be flanked with Xbal(underlined in primer EV3643) and MluI (underlined in primer EV3644)sites by PCR with:

primer EV3643 (SEQ ID No 31)

and primer EV3644 (SEQ ID No 32) (CACTACGCGTAAAGGTGAGGAAGTATATG).

Primer EV3643 contains an additional Clal site (dotted line) followingthe Xbal site. pEVE2798 served as template (FIG. 25).

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. The PCR wasaccomplished with an initial denaturation step of 30 sec at 98° C.followed by 30 cycles with 10 sec at 98° C./10 sec at 54° C./25 sec at72° C., and a final extension step of 5 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.). The 1.3 kb nat1 expression cassette was restriction digestedwith MluI and Xbal enzymes (New England Biolabs, Ipswich, Mass.) andligated for 2 h at room temperature to the 2.6 kb vector backbone ofpEVE2787 (FIG. 23) linearized with MU and Xbal enzymes (New EnglandBiolabs, Ipswich, Mass.) and gel-purified with the Zymoclean™ Gel DNARecovery Kit (Zymo Research Corporation, Irvine, Calif.) using T4 DNAligase (New England Biolabs, Ipswich, Mass.) (FIG. 26).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2852 (FIG. 27) contains the nat1 selectionmarker under the control of the ribulose reductase (poRR) promoter andorotidine-5′-phosphate decarboxylase (poURA3) terminator and flanked bytwo loxP sites.

The integration plasmid does not contain any P. ohmeri homologousfragments needed for site specific integration into the genome, so far.This sites were attached in the next steps.

The 5′ homologous region upstream of be LEU2 open reading frame wasamplified from 50 ng poARS vector (FIG. 6) with:

primer EV3548 (SEQ ID No 33) (CACTCTGCAGGATCCAAGCTATCAACGAGA)containing a PstI site (underlined) and primer EV3549 (SEQ ID No 34)(CACTGCATGCGTTGCGGAAAAAACAGCC) containing a SphI site (underlined).

The PCR was performed in a reaction mix consisting of 200 μM of eachdNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. Theamplification was accomplished with an initial denaturation step of 30sec at 98° C. followed by 30 cycles with 10 sec at 98° C./10 sec at 61°C./15 sec at 72° C., and a final extension step of 5 minutes at 72° C.The PCR product was separated on a 1% agarose gel, extracted andpurified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.). The 567 bp fragment was restrictiondigested with PstI and SphI enzymes (New England Biolabs, Ipswich,Mass.) and ligated for 2 h at room temperature to the 3.9 kb vectorbackbone of pEVE2852 (FIG. 27) linearized with PstI and SphI restrictionenzymes (New England Biolabs, Ipswich, Mass.) and gel-purified withZymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.) using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG.29).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2855 (FIG. 28) contains a fragment homologousto the 5′ region upstream of the LEU2 open reading frame and a nat1marker flanked by two loxP sites.

The 3′ homologous region downstream of be LEU2 open reading frame wasamplified from 50 ng poARS vector (FIG. 6) with:

primer EV3550 (SEQ ID No 35) (CACT CCATGG AGTAGGTATATAAAAATATAAGAG)containing a NcoI site (underlined) and primer EV3551 (SEQ ID No 36)(CACTGCTAGCGTCGACAACAGCAACTAG) containing a NheI site (underlined).

The PCR was performed in a reaction mix consisting of 200 μM of eachdNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. Theamplification was accomplished with an initial denaturation step of 30sec at 98° C. followed by 30 cycles with 10 sec at 98° C./10 sec at 51°C./25 sec at 72° C., and a final extension step of 5 minutes at 72° C.The PCR product was separated on a 1% agarose gel, extracted andpurified using the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.). The 1.3 kb fragment was restrictiondigested with NcoI and NheI enzymes (New England Biolabs, Ipswich,Mass.) and ligated for 2 h at room temperature to the 4.4 kb vectorbackbone of pEVE2855 (FIG. 28) linearized with NcoI and NheI restrictionenzymes (New England Biolabs, Ipswich, Mass.) and gel-purified withZymaclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.) using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG.29).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting final LEU2 deletion plasmid pEVE2864 (FIG. 30) contains afragment homologous to the 5′ region upstream and a fragment homologousto the 3′ region downstream of the LEU2 open reading frame and a nat1marker flanked by two loxP sites.

Example 16 Generation of a Mutant P. ohmeri Strain Auxotrophic forLeucine

Since the generated P. ohmeri CNCM I-4605 strain did not display anyauxotrophy so far, a LEU2 open reading frame deletion was performed, soas to be able to use the LEU2 selection marker for gene integrations.

For this purpose plasmid pEVE2864 (FIG. 30) was restriction digestedwith EcoRV and PstI enzymes (New England Biolabs, Ipswich, Mass.) for2.5 h at 37° C. and the mixture used to transform the Mut165 strainaccording to the procedure described in Example 12.

To the regenerated cells, 7 ml of 50° C. warm top agar (1% yeastextract, 2% peptone, 2% glucose, 1 M sorbitol, pH 5.8 and 2.5% Nobleagar) with 25 μg/ml natamycin was added and the mixture was pouredevenly onto pre-warmed, sorbitol containing selection plates (1% yeastextract, 2% peptone, 2% glucose, 1 M sorbitol. pH 5.8 and 2% agar) with25 μg/ml natamycin. Plates were incubated for 4 days at 30° C. Deletionof the LEU2 open reading frame was verified by no growth on selectiveplates without leucine and confirmed by colony PCR using:

primer EV3393 (SEQ ID No 29) (CAAGCATGCGGGAATGATAAGAGACTTTG) andprimer EV3795 (SEQ ID No 37) (CAAGTCGTGGAGATTCTGC)

The 1.6 kb fragment was amplified with an initial denaturation step of30 sec at 98° C. followed by 30 cycles with 10 sec at 98° C./10 sec at51° C./25 sec at 72° C., and a final extension step of 5 minutes at 72°C.

The resulting strain contains the full open reading frame deletion ofthe LEU2 gene in a CNCM I-4605 background and was deposited in France onFeb. 5, 2015, with the Collection Nationale de Cultures deMicroorganismes [National Collection of Microorganism Cultures] of theInstitut Pasteur (CNCM), 25 rue du Docteur Roux, 75724 PARIS codex 15,under number I-4955.

Example 17. Construction of a Double Expression Plasmids Comprising theNADPH-Specific Xylitol Dehydroctenase of P. stipitis and theNAD+-Specific D-Arabitol 4-Oxidoreductase of E. coli

In order to be able to express the NADPH-specific xylitol dehydrogenaseof P. stipitis and the NAD specific D-arabitol 4-oxidoreductase of E.coli in the mutant P. ohmeri strain only auxotrophic for leucine,construction of a double expression plasmid was required.

The expression cassette containing the NADPH-specific xylitoldehydrogenase of P. stipitis was released from pEVE2562 (FIG. 12) byrestriction cutting with SpeI and SacII enzymes (New England Biolabs,Ipswich, Mass.). The 1.9 kb fragment was gel-purified using Zymoclean™Gel DNA Recovery Kit (Zymo Research Corporation, Irvine, Calif.) andblunted with the Blunting Enzyme Mix kit (New England Biolabs, Ipswich,Mass.) for 15 min at room temperature, followed by heat inactivation ofthe enzymes for 10 min at 70° C. The insert was then ligated for 2 h atroom temperature to the 12.1 kb SpeI-linearized, blunted,dephosphorylated (1 h at 37° C. using Antarctic phosphatase—New EnglandBiolabs, Ipswich, Mass.) and gel-purified pEVE3157 backbone (FIG. 21)containing the NAD+-specific D-arabitol 4-oxidoreductase of E. coliusing T4 DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG. 31).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3318 (FIG. 32) contains the double expressionconstruct of the NADPH-specific xylitol dehydrogenase of P. stipitisflanked by a P. ohmeri ribulose reductase promoter and terminator (poRR)and the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli under thecontrol of the P. ohmeri phosphoglycerate kinase (poPGK1) promoter andribulose reductase (poRR) terminator and the poLEU2 selection marker.

Example 18. Construction of Integrative Vectors for the Emission of theE. coli NAD⁺-Specific D-arabitol 4-Oxidoreductase Gene and the P.stipitis NADPH-Specific Xylitol Dehydrogenase Gene in P. ohmeri

The NAD⁺-specific D-arabitol 4-oxidoreductase gene of E. coli and theNADPH-specific xylitol dehydrogenase gene of P. stipitis shouldultimately become an integral part of the P. ohmeri genome. Therefore,an integrative vector with a LEU2 selection marker had to beconstructed, by replacing the nat1 selection marker of pEVE2852 andincorporating the double expression construct of arabitol oxidoreductaseand xylitol dehydrogenase.

For this purpose, the P. ohmeri LEU2 open reading frame, flanked by anAscI and SphI sites, was generated by PCR with:

primer EV3645 (SEQ ID No 38) (CAAGGCGCGCCAAAATGTCTACCAAAACCATTAC) andprimer EV3646 (SEQ ID No 39) (GGAGCATGCCTACTTTCCCTCAGCCAAG).

Amplification was performed with 50 ng of poARS (FIG. 6) template in areaction mix consisting of 200 μM of each dNTP and 0.5 μM of each primerwith 0.02 U/μl of iProof™ polymerase (BIO-RAD, Hercules, Calif.) in theappropriate 1× buffer. The PCR was accomplished with an initialdenaturation step of 30 sec at 98° C. followed by 30 cycles with 10 secat 98° C./10 sec at 57° C./20 sec at 72° C., and a final extension stepof 5 minutes at 72° C. The PCR product was separated on a 1% agarosegel, extracted and purified using the Zymoclean™ Gel DNA Recovery Kit(Zymo Research Corporation, Irvine, Calif.). The amplified LEU2 openreading frame was subsequently restriction digested with AscI and SphIenzymes (New England Biolabs, Ipswich, Mass.).

Additionally, a blunting of the SphI site with the Blunting Enzyme Mixkit (New England Biolabs, Ipswich, Mass.) for 15 min at roomtemperature, followed by heat inactivation of the enzymes for 10 min at70° C. was performed in between the SphI and AscI digestion. The 1.1 kbgel-purified fragment was than ligated to the gel-purified 11 kb vectorbackbone of pEVE2811 cut with SphI and AscI restriction enzymes (NewEngland Biolabs, Ipswich, Mass.). Also the SphI site of the vector wasblunted for 15 min at room temperature with the Blunting Enzyme Mix kit(New England Biolabs, Ipswich, Mass.), followed by a heat inactivationstep of 10 min at 70° C. before the digestion with AscI was performed.Additionally, the vector was dephosphorylated for 1 h at 37° C. usingAntarctic phosphatase (New England Biolabs, Ipswich, Mass.). Theligation of the LEU2 open reading frame and the vector backbone wasperformed for 2 h at room temperature using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2862 (FIG. 33) contains the P. ohmeri LEU2marker flanked by a P. ohmeri ribulose reductase (poRR) promoter and anorotidine-5′-phosphate decarboxylase (poURA3) terminator.

Subsequently, the LEU2 marker was amplified by PCR using:

primer EV3643 (SEQ ID No 31) (CACTATCGATGGATCCGTAGAAATCTTG)containing a ClaI site and primer EV3644 (SEQ ID No 32)(CACTACGCGTAAAGGTGAGGAAGTATATG) containing a MluI site (underline) andpEVE2862 (FIG. 33) as template.

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. The PCR wasaccomplished with an initial denaturation step of 30 sec at 98° C.followed by 30 cycles with 10 sec at 98° C./10 sec at 54° C./30 sec at72° C., and a final extension step of 5 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.). The amplified 1.8 kb long LEU2 fragment was restrictiondigested with Clal and MluI enzymes (New England Biolabs, Ipswich,Mass.) and ligated for 2 h at room temperature to the 2.6 kb Clal andMluI (New England Biolabs, Ipswich, Mass.) restriction digested andgel-purified vector backbone of pEVE2852 (FIG. 27) using T4 DNA ligase(New England Biolabs, Ipswich, Mass.) (FIG. 34).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE2865 (FIG. 35) contains the P. ohmeri LEU2marker flanked by two loxP sites.

For cloning of the integration vector, pEVE2865 was restriction digestedwith SalI enzyme (New England Biolabs, Ipswich, Mass.), blunted with theBlunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.) for 15 minat room temperature, followed by heat inactivation of the enzymes for 10min at 70° C. and dephosphorylated dephosphorylated for 1 h at 37° C.using Antarctic phosphatase (New England Biolabs, Ipswich, Mass.).

The 4.5 kb gel-purified fragment of the vector backbone was used forligation. As insert served a double expression construct of theNADPH-specific xylitol dehydrogenase genes of P. stipitis and theNAD⁺-specific D-arabitol 4-oxidoreductase of E. coli released frompEVE3318 (FIG. 32) by restriction cutting with NdeI and SacII enzymes(New England Biolabs, Ipswich, Mass.).

The 4.4 kb fragment was gel-purified using Zymoclean™ Gel DNA RecoveryKit (Zymo Research Corporation, Irvine, Calif.) and blunted with theBlunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.) for 15 minat room temperature, followed by heat inactivation of the enzymes for 10min at 70° C., followed by an additional gel purification. The vectorbackbone of pEVE2865 and the insert of pEVE3318 were ligated for 2 h atroom temperature using T4 DNA ligase (New England Biolabs, Ipswich,Mass.) (FIG. 34).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3387 (FIG. 36) contains the double expressionconstruct of the NADPH-specific xylitol dehydrogenase gene of P.stipitis flanked by a P. ohmeri ribulose reductase promoter andterminator (poRR) and the NAD⁺-specific D-arabitol 4-oxidoreductase ofE. coli under the control of the P. ohmeri phosphoglycerate kinase(poPGK1) promoter and transketolase (poTKL) terminator. As selectionmarker serves a P. ohmeri LEU2 gene flanked by two loxP sites.

Example 19. Construction of a First Generation Integrative P. ohmeriStrain Secreting Xylitol into the Media

The previously described vector was used to randomly integrate theNAD⁺-specific D-arabitol 4-oxidoreductase gene of E. coli and theNADPH-specific xylitol dehydrogenase gene of P. stipitis into the genomeof P. ohmeri.

For this purpose strain CNCM I-4955 (Example 16) auxotrophic for leucinewas transformed with pEVE3387 (FIG. 36) restriction digested with NotI(New England Biolabs, Ipswich, Mass.) for 3 h at 37° C. according to theprocedure described in Example 12. Transformants were selected onsorbitol plates without any leucine.

The resulting strain contains the NAD⁺-specific D-arabitol4-oxidoreductase gene of E. coli and the NADPH-specific xylitoldehydrogenase gene of P. stipitis randomly integrated into the P. ohmerigenome and was deposited in France on May 20, 2015, with the CollectionNationale de Cultures de Microorganismes [National Collection ofMicroorganism Cultures] of the Institut Pasteur (CNCM), 25 rue duDocteur Roux, 75724 Cedex 15, under number I-4982.

Example 20. Construction of a Double/Triple Expression PlasmidComprising the NADPH-Specific Xylitol Dehydrogenase of G. oxydans andthe NAD⁺-Specific D-Arabitol 4-Oxidoreductase of E. coli

In order to be able to express the NADPH-specific xylitol dehydrogenaseof G. oxydans and the NAD⁺-specific D-arabitol 4-oxidoreductase of E.coli in the mutant P. ohmeri strain only auxotrophic for leucine,construction of a double expression plasmid was required.

The expression cassette containing the NADPH-specific xylitoldehydrogenase of G. oxydans was released from pEVE3284 (FIG. 10) byrestriction cutting with SpeI and SacII enzymes (New England Biolabs,Ipswich, Mass.). The 1.6 kb fragment was gel-purified using Zymoclean™Gel DNA Recovery Kit (Zymo Research Corporation, Irvine, Calif.) andblunted with the Blunting Enzyme Mix kit (New England Biolabs, Ipswich,Mass.) for 15 min at room temperature, followed by heat inactivation ofthe enzymes for 10 min at 70° C. The vector backbone used consisted ofthe 12.1 kb SpeI-linearized (New England Biolabs, Ipswich, Mass.) andgel-purified (Zymoclean™ Gel DNA Recovery Kit—Zymo Research Corporation,Irvine, Calif.) pEVE3157 backbone (FIG. 21) containing the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli.

The backbone has additionally been blunted for 15 min at roomtemperature with the Blunting Enzyme Mix kit (New England Biolabs,Ipswich, Mass.), followed by heat inactivation of the enzymes for 10 minat 70° C. and dephosphorylated for 1 h at 37° C. using Antarcticphosphatase (New England Biolabs, Ipswich, Mass.). Ligation wasperformed for 2 h at room temperature using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass.) (FIG. 37).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmids pEVE3322 and pEVE3324 (FIG. 38) contain eitherthe double expression construct of the NADPH-specific xylitoldehydrogenase of G. oxydans flanked by a P. ohmeri ribulose reductasepromoter and terminator (poRR) and the NAD⁺-specific D-arabitol4-oxidoreductase of E. coli under the control of the P. ohmeriphosphoglycerate kinase (poPGK1) promoter and transketolase (poTKL)terminator or the triple expression construct of two NADPH-specificxylitol dehydrogenase genes of G. oxydans flanked by a P. ohmeriribulose reductase promoter and terminator (poRR) and the NAD⁺-specificD-arabitol 4-oxidoreductase of E. coli under the control of the P.ohmeri phosphoglycerate kinase (poPGK1) promoter and transketolase(poTKL) terminator and the poLEU2 selection marker.

Example 21. Construction of Integrative Vectors for, the Expression ofthe E. coli NAD⁺-Specific D-Arabitol 4-Oxidoreductase Gene and the G.oxydans NADPH-Specific Xylitol Gene in P. ohmeri

Besides the integrative vector containing the NADPH-specific xylitoldehydrogenase of P. stipitis and the NAD⁺-specific D-arabitol4-oxidoreductase gene of E. coli also plasmids containing theNADPH-specific xylitol dehydrogenase of G. oxydans were generated.

For this purpose, the double and triple expression cassettes containingeither one or two NADPH-specific xylitol dehydrogenase of G. oxydans andthe NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli were releasedfrom pEVE3322 and pEVE3324 (FIG. 38) respectively, by restrictioncutting with NdeI and SacII enzymes (New England Biolabs, Ipswich,Mass.).

The 4.1 kb and 5.7 kb fragments were gel-purified using Zymoclean™ GelDNA Recovery Kit (Zymo Research Corporation, Irvine, Calif.) and bluntedwith the Blunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.)for 15 min at room temperature, followed by heat inactivation of theenzymes for 10 min at 70° C. As vector served the gel-purified(Zymoclean™ Gel DNA Recovery Kit—Zymo Research Corporation, Irvine,Calif.), 5.7 kb SalI-linearized pEVE2865 (FIG. 35).

The vector backbone has additionally been blunted for 15 min at roomtemperature with the Blunting Enzyme Mix kit (New England Biolabs,Ipswich, Mass.), followed by heat inactivation of the enzymes for 10 minat 70° C. and dephosphorylation for 1 h at 37° C. using Antarcticphosphatase (New England Biolabs, Ipswich, Mass.). Ligation of vectorand insert was performed for 2 h at room temperature to using T4 DNAligase (New England Biolabs, Ipswich, Mass.) (FIG. 39).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmids pEVE3390 and pEVE3392 (FIG. 40) contain thedouble or triple expression constructs of either one or twoNADPH-specific xylitol dehydrogenase genes of G. oxydans flanked by a P.ohmeri ribulose reductase promoter and terminator (poRR) and theNAD⁺-specific D-arabitol 4-oxidoreductase of E. coli under the controlof the P. ohmeri phosphoglycerate kinase (poPGK1) promoter andtransketolase (poTKL) terminator. As selection marker serves a P. ohmeriLEU2 gene flanked by two loxP sites.

Example 22. Construction of Second Generation Integrative StrainsCapable of Secreting More than 100 g/L Xylitol

First generation strain CNCM I-4982 containing a randomly integratedcopy of the NAD⁺-specific D-arabitol 4-oxidoreductase gene of E. coliand the NADPH-specific xylitol dehydrogenase gene of P. stipitis wasused to further integrate additional copies of the two heterologousenzymes.

However, in order to be able to integrate above constructs the LEU2selection marker had to be removed. For this purpose first generationstrain CNCM I-4982 was transformed with vector pEVE3163 according to theprocedure described in Example 12. The vector pEVE3163 contains the CRErecombinase of bacteriophage P1 (codon optimized according to Table 7)flanked by a P. ohmeri ribulose reductase promoter and terminator(poRR). Removal of the LEU2 selection marker was confirmed by no-growthof clones on plates without leucine.

The resulting strain EYS3842 was transformed with pEVE3390 or pEVE3392(FIG. 40) restriction digested with NotI (New England Biolabs, Ipswich,Mass.) for 3 h at 37° C. according to the procedure described in Example12. Transformants were selected on sorbitol plates without any leucine.

Resulting second generation strain EYS3929 contains two NAD⁺-specificD-arabitol 4-oxidoreductase genes of E. coli and two NADPH-specificxylitol dehydrogenase genes, one from G. oxydans and a second one fromP. stipitis randomly integrated into the genome. Strain EYS3930, on theother hand, contains an additional NADPH-specific xylitol dehydrogenasegene of G. oxydans.

Example 23. Construction of a Further Vector Used for the Integration ofAdditional Gene Copies of the NAD⁺-Specific D-Arabitol 4-Oxidoreductaseof E. coli and the NADPH-Specific Xylitol Dehydrogenase of G. oxydans

In order to construct a further integration vector, a double expressioncassette of the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli andthe NADPH-specific xylitol dehydrogenase of G. oxydans was amplified byPCR using:

primer EV4904 (SEQ ID No 40) (ATATCCCGGGCACCGTCATCACCGAAACGC)containing a SmaI site and primer EV4905 (SEQ ID No 41)(ATATCCCGGGCACGACCACGCTGATGAGC) containing a SmaI site (underline) andpEVE3321 as template

Amplification was performed in a reaction mix consisting of 200 μM ofeach dNTP and 0.5 μM of each primer with 0.02 U/μl of iProof™ polymerase(BIO-RAD, Hercules, Calif.) in the appropriate 1× buffer. The PCR wasaccomplished with an initial denaturation step of 30 sec at 98° C.followed by 30 cycles with 10 sec at 98° C./10 sec at 68° C. 175 sec at72° C., and a final extension step of 5 minutes at 72° C. The PCRproduct was separated on a 1% agarose gel, extracted and purified usingthe Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.).

The amplified 3.9 kb long fragment was restriction digested with Smal(New England Biolabs, Ipswich, Mass.) and ligated for 2 h at roomtemperature to the 4.4 kb Pvull (New England Biolabs, Ipswich, Mass.)linearized, Antarctic phosphatase (New England Biolabs, Ipswich, Mass.)dephosphorylated and gel-purified vector backbone of pEVE2865 (FIG. 35)using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG. 41).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmids pEVE4390 (FIG. 42) contains the double expressionconstruct of the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coliunder the control of the P. ohmeri phosphoglycerate kinase (poPGK1)promoter and transketolase (poTKL) terminator and the NADPH-specificxylitol dehydrogenase gene of G. oxydans flanked by a P. ohmeri ribulosereductase promoter and terminator (poRR). As selection marker serves aP. ohmeri LEU2 gene flanked by two loxP sites.

Example 24. Construction of a Vector Used for the Integration of theNADPH-Specific Xylitol Dehydrogenase of G. oxydans and the NAD⁺-SpecificD-Arabitol 4-Oxidoreductase of R. solanacearum

An additional integrative vector for the expression of theNADPH-specific xylitol dehydrogenase of G. oxydans and of theNAD+-specific D-arabitol 4-oxidoreductase of R. solanacearum wasconstructed as follows: In a first step a double expression vectorcontaining the two above genes was generated, This double expressioncassette was the cloned into an integrative loxP vector.

A DNA fragment encoding the NAD+-specific D-arabitol 4-oxidoreductasegene of Ralstonia solanacearum was chemically synthesized by GeneArt®Gene Synthesis (Life Technologies, Regensburg, Germany) according to thesubmitted sequence of sequence SEQ ID No 42.

Nucleotides 2310548 to 2309151 of sequence AL646052.1 (obtained from theNCBI GenBank database) coding for the dalD gene were used as templateand subjected to codon optimization for use in P. ohmeri ATCC 20209according to Table 7 (above), using the Optimizer program. At the 5′ and3′ ends of the resulting sequence, nucleotides encoding for therecognition sites of the restriction enzymes AscI (GGCGCGCC) and SphI(GCATGC) respectively, were manually added in the text file, in order tofacilitate further cloning. Additionally, an adenosine triplet wasincluded in front of the start ATG to account for an adenosine at the −3position in the Kozak-like sequence of yeasts.

The final sequence (SEQ ID No 42) was then submitted for synthesis toGeneArt (Regensburg, Germany). The synthesized DNA fragment encoding thedalD gene was delivered as 5 μg lyophilized plasmid DNA in a pMA-RQderived vector (13AB2EGP, FIG. 43).

The 1.4 kb fragment of the D-arabitol 4-oxidoreductase from R.solanacearum was released from vector 13AB2EGP (FIG. 43) by restrictiondigested with AscI and SphI (New England Biolabs, Ipswich, Mass.) andgel-purified with the Zymoclean™ Gel DNA Recovery Kit (Zymo ResearchCorporation, Irvine, Calif.). The insert was then ligated with the 11.8kb backbone of pEVE2560 (FIG. 8) linearized with AscI and SphI (NewEngland Biolabs, Ipswich, Mass.) and gel purified using T4 DNA ligase(New England Biolabs, Ipswich, Mass.) (FIG. 44).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE3898 (FIG. 45) contains the codon-optimised R.solanacearum NAD+-specific D-arabitol 4-oxidoreductase flanked by aribulose reductase promoter and terminator of P. ohmeri and the poLEU2selection marker.

In a next step the expression cassette containing the NADPH-specificxylitol dehydrogenase of G. oxydans flanked by a phosphoglycerate kinasepromoter (poPGK) and ribulose reductase terminator (poRR) was releasedfrom pEVE3960 by restricon digest with SpeI and SacII (New EnglandBiolabs, Ipswich, Mass.). The 1.8 kb fragment was gel-purified usingZymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine,Calif.) and blunted with the Blunting Enzyme Mix kit (New EnglandBiolabs, Ipswich, Mass.) for 15 min at room temperature, followed byheat inactivation of the enzymes for 10 min at 70° C. As vector servedthe gel-purified (Zymoclean™ Gel DNA Recovery Kit—Zymo ResearchCorporation, Irvine, Calif.), 13.2 kb SalI-linearized pEVE3898. Thevector backbone has additionally been blunted for 15 min at roomtemperature with the Blunting Enzyme Mix kit (New England Biolabs,Ipswich, Mass.), followed by heat inactivation of the enzymes for 10 minat 70° C. and dephosphorylation for 1 h at 37° C. using Antarcticphosphatase (New England Biolabs, Ipswich, Mass.). Ligation of vectorand insert was performed for 2 h at room temperature to using T4 DNAligase (New England Biolabs, Ipswich, Mass.) (FIG. 44).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE4077 (FIG. 46) contains the double expressionconstruct of the NADPH-specific xylitol dehydrogenase of G. oxydansflanked by a P. ohmeri phosphoglycerate kinase promoter (poPGK) and aribulose reductase terminator (poRR) and the NAD-specific D-arabitol4-oxidoreductase of R. solanacearum under the control of the P. ohmeriribulose reductase promoter and (poRR) terminator and the poLEU2selection marker.

Finally, the double expression cassette of the NADPH-specific xylitoldehydrogenase of G. oxydans and the NAD⁺-specific D-arabitol4-oxidoreductase of R. solanacearum was released from pEVE4077 (FIG. 46)by restriction cutting with SapI (New England Biolabs, Ipswich, Mass.).The 5.9 kb fragment was gel-purified using Zymoclean™ Gel DNA RecoveryKit (Zymo Research Corporation, Irvine, Calif.) and blunted with theBlunting Enzyme Mix kit (New England Biolabs, Ipswich, Mass.) for 15 minat room temperature, followed by heat inactivation of the enzymes for 10min at 70° C. As vector served the gel-purified (Zymoclean™ Gel DNARecovery Kit—Zymo Research Corporation, Irvine, Calif.), 4.4 kbEcoRV-linearized pEVE2865 (FIG. 35), dephosphorylated for 1 h at 37° C.with Antarctic phosphatase (New England Biolabs, Ipswich, Mass.).Ligation of vector and insert was performed for 2 h at room temperatureto using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) (FIG. 44).

After transformation of XL10 Gold ultracompetent cells (AgilentTechnologies, Santa Clara, Calif.) with the ligation mixture, plasmidDNA was isolated using the Zyppy™ Plasmid Miniprep Kit (Zymo ResearchCorporation, Irvine, Calif.) and further characterized by restrictiondigestion and sequencing (Microsynth, Balgach, Switzerland).

The resulting plasmid pEVE4377 (FIG. 47) contains the double expressionconstruct of the NADPH-specific xylitol dehydrogenase of G. oxydans andthe NAD⁺-specific D-arabitol 4-oxidoreductase of R. solanacearum and thepoLEU2 selection marker flanked by two loxP sites.

Example 25. Construction of Third Generation Integrative Strains withIncreased Productivity of Xylitol

The LEU2 marker of second generation strains EYS3929 and EYS3930(Example 22) was loxed out as described in Example 18 using vectorpEVE3163. The resulting strains EYS4118 and EYS4119 were transformedwith pEVE4377 (FIG. 47) and pEVE4390 (FIG. 42), respectively, Thevectors were restriction digested with NotI (New England Biolabs,Ipswich, Mass.) for 3 h at 37° C. according to the procedure describedin Example 12. Transformants were selected on sorbitol plates withoutany leucine.

Resulting third generation strain EYS4353 contains three NAD⁺-specificD-arabitol 4-oxidoreductase genes, two from E. coli and one from R.solanacearum and three NADPH-specific xylitol dehydrogenase genes, twofrom G. oxydans and one from P. stipitis randomly integrated into thegenome.

The second third generation strain, on the other hand, contains threecopies of the NAD⁺-specific D-arabitol 4-oxidoreductase of E. coli,three copies of the NADPH-specific xylitol dehydrogenase of G. oxydansand one copy from P. stipitis, background and was deposited in France onMar. 5, 2015, with the Collection Nationale de Cultures deMicroorganismes [National Collection of Microorganism Cultures] of theInstitut Pasteur (CNCM), 25 rue du Docteur Roux, 75724 PARIS Cedex 15,under number I-4960.

Example 26. Construction of Fourth Generation Integrative Strains

The LEU2 marker of third generation strains CNCM I-4960 (Example 25) wasloxed out as described in Example 18 using vector pEVE3163. Theresulting strain EYS4955 was transformed with pEVE4377 (FIG. 47)restriction digested with NotI (New England Biolabs, Ipswich, Mass.) for3 h at 37° C. according to the procedure described in Example 12.Transformants were selected on sorbitol plates without any leucine.

Resulting fourth generation strain contains four NAD⁺-specificD-arabitol 4-oxidoreductase genes, three from E. coli and one from R.solanacearum and four NADPH-specific xylitol dehydrogenase genes, threefrom G. oxydans and one from P. stipitis randomly integrated into thegenomeand was deposited in France on May 20, 2015, with the CollectionNationale de Cultures de Microorganismes [National Collection ofMicroorganism Cultures] of the Institut Pasteur (CNCM), 25 rue duDocteur Roux, 75724 PARIS Cedex 15, under number I-4981.

Example 27. Polyol Production with Pichia ohmeri Strains (SyntheticMedium)

The yeast strains CNCM I-4605, CNCM I-4982. CNCM I-4960 & CNCM I-4981constructed as described above, were fermented according to thefollowing protocol.

The fermentation process is run under Nitrogen-limitation and can beseparated into a growth phase and a production phase. During the growthphase the ammonia in the medium is completely consumed to producebiomass, once the biomass formation stops the production phase startsand Polyol levels increase. The platform used for the describedfermentation process was a Multifors 2 from INFORS HT, using vesselswith a working volume of 1 L. The fermenters were equipped with twoRushton six-blade disc turbines. Air was used for sparging thefermenters.

Temperature, pH, agitation, and aeration rate were controlled throughoutthe cultivation. The temperature was maintained at 36° C. The pH waskept at 3 by automatic addition of 5M KOH.

The aeration rate was kept at 1.0 vvm and the initial stirrer speed wasset to 300 rpm. In order to prevent the Dissolved Oxygen (DO) to dropbelow 20% an automatic stirring cascade was employed. The operatingconditions used in the fermentation process are summarized in Table 10.

TABLE 10 Operating conditions for the Polyol production fermentationsParameter Set-point Volume of liquid [L] 1 Temperature [° C.] 36  pH 3Agitation speed [rpm] Initially 300, then DO setpoint (20%) controlledstirrer cascade Air flow rate [vvm] 1

For inoculation of the fermenters a 1-stage propagation culture wasused. The composition of the used propagation culture medium isdescribed in table 11, Propagation cultures were prepared by inoculating100 ml of medium in a 500-ml shake flask with 4 baffles (indent), Theshake flasks were incubated on a shaking table at 30° C. and 150 rpm.The cells were grown for ˜24 hrs into mid-exponential phase.

TABLE 11 Propagation culture medium composition. Raw materialConcentration [g/L] Glucose monohydrate C₆H₁₂O₆ * H₂O 46 Antifoam Erol18 1 drop Potassium dihydrogenphosphate KH₂PO₄ 6 Magnesium sulfateheptahydrate MgSO₄ * 7H₂O 2.4 Ammonium sulfate (NH₄)₂SO₄ 0.16 Iron(II)ammonium sulfate hexahydrate Fe(SO₄)₂(NH₄)₂ * 6H₂O 0.012 Manganese (II)sulfate monohydrate MnSO₄ * H₂O 0.0007 Zinc sulfate heptahydrate ZnSO₄ *7H₂O 0.00007 Biotine C₁₀H₁₆N₂O₃S 0.0004 Sodium phosphate Na₂HPO₄ 0.292Citric acid monohydrate C₆H₈O₇ * H₂O 0.835

Prior to inoculation, an amount of the medium in the fermenterequivalent to the amount of inoculum was removed and an aliquot of thepropagation culture was used for inoculation of the fermenter to a finalvolume of 1 L and an OD₆₀₀-at-start of ca. 0.2 (CDW ca. 0.03 g/L). Thecomposition of the medium used in the fermenter is described in table12.

TABLE 12 Fermentation medium composition. Raw material Concentration[g/L] Glucose monohydrate C₆H₁₂O₆ * H₂O 250 Antifoam Erol 18 0.67Potassium dihydrogenphosphate KH₂PO₄ 6 Magnesium sulfate heptahydrateMgSO₄ * 7H₂O 2.4 Ammonium sulfate (NH₄)₂SO₄ 4 Iron(II) ammonium sulfatehexahydrate Fe(SO₄)₂(NH₄)₂ * 6H₂O 0.012 Manganese (II) sulfatemonohydrate MnSO₄ * H₂O 0.0007 Zinc sulfate heptahydrate ZnSO₄ * 7H₂O0.00007 Biotine C₁₀H₁₆N₂O₃S 0.0004

Samples were withdrawn in regular intervals and the total fermentationbroth was analyzed for Glucose consumption and extracellular Polyol(Xylitol, Arabitol and Ribitol) formation. Furthermore commonfermentation metabolites (Glycerol, Acetate, Ethanol, Pyruvate, Malate,Fumarate & Succinate) were determined. The increase in biomass was onone hand followed by OD₆₀₀ and on the other hand by cell dry weight(CDW) determination. The above mentioned measurements were used todetermine Polyol production, Arabitol or Xylitol yield and productivity;the results are shown in table 13.

TABLE 13 Polyol production with Pichia ohmeri strains (syntheticmedium). CNC CNCM CNCM I- CNCM I-4605 I-4982 4960 I-4981 ElapsedFermentation 67 79 146 64 66 Time (EFT) [h] Glucose [g/L] 0 0 0 0 0Arabitol [g/L] 118 74 0 0 0 Ribitol [g/L] 0 6 2 7 5 Xylitol [g/L] 0 2860 110 120 Yield Arabitol [%] 52 — — — — Yield Xylitol [%] — 12 26 44 48Productivity [g/L/h] 1.76 0.35 0.41 1.71 1.81

Pichia ohmeri CNCM I-4605 produces arabitol only.

Pichia ohmeri CNCM I-4982 produces arabitol, xylitol and ribitol. Inthis strain one copy of NAD⁺-D-arabitol 4-oxidoreductase gene and onecopy of NADPH-specific xylitol dehydrogenase gene have been integrated.The modified strain is now able to consume arabitol. Consequently, aftertotal consumption of glucose, arabitol and ribitol are re-consumed byCNCM I-4982 to produce more xylitol.

Pichia ohmeri CNCM I-4960 (third generation) and CNCM I-4981 (fourthgeneration) produce xylitol and ribitol but no more arabitol, Theintracellular conversion of arabitol in xylulose and xylitol isefficient enough to avoid the excretion of arabitol into the broth. Themore copies of the genes encoding for the NAD-specific D-arabitoloxidoreductase and the NADPH-specific xylitol dehydrogenase have beenintroduced into P. ohmeri, the higher are the titer, yield andproductivity of xylitol.

The invention claimed is:
 1. A method for producing xylitol, the methodcomprising: culturing in a culture medium a recombinant Pichia ohmericapable of producing a xylitol titer of at least 15 g/L, in asupernatant within 48 h of culture, the recombinant Pichia ohmericomprising: a heterologous nucleic acid sequence encoding aNAD+-specific D-arabitol 4-oxidoreductase (EC 1.1.1.11) from Escherichiacoli and/or Ralstonia solanacearum comprising the amino acid sequence ofSEQ ID NO: 2 and/or 43 using D-arabitol as a substrate and producingD-xylulose as a product; and a heterologous nucleic acid sequenceencoding a NADPH-specific xylitol dehydrogenase from Pichia stipitisand/or Gluconobacter oxydans comprising the amino acid sequence of SEQID NO: 5 and/or 8 using D-xylulose as a substrate and producing xylitolas a product; and recovering the produced xylitol.
 2. The methodaccording to claim 1, wherein the culture medium provides therecombinant Pichia ohmeri with a carbon source.
 3. The method accordingto claim 2, wherein the carbon source includes D-glucose.
 4. The methodaccording to claim 1, wherein the host cell recombinant Pichia ohmeriproduces D-arabitol from D-glucose.
 5. The method according to claim 1,wherein the recombinant Pichia ohmeri produces D-arabitol from D-glucoseunder a high osmotic pressure medium.
 6. The method according to claim1, wherein the recombinant Pichia ohmeri does not consume D-arabitol asa sole carbon source.
 7. The method according to claim 1, wherein thesequence encoding the NAD+-specific D-arabitol 4-oxidoreductase (EC1.1.1.11) comprises SEQ ID NO:3 or SEQ ID NO:42.
 8. The method accordingto claim 1, wherein the sequence encoding the NADPH-specific xylitoldehydrogenase comprises SEQ ID NO:6 or SEQ ID NO:9.
 9. The methodaccording to claim 1, wherein the host cell recombinant Pichia ohmeri isa strain selected from strains 1-4982, 1-4960, and 1-4981 deposited atthe National Collection of Microorganism Cultures.
 10. The methodaccording to claim 1, wherein the recombinant Pichia ohmeri comprisestwo, three, or four sequences encoding a NAD+-specific D-arabitol4-oxidoreductase and/or two, three, or four sequences encoding theNADPH-specific xylitol dehydrogenase.
 11. The method according to claim1, further comprising purifying the recovered xylitol.
 12. The methodaccording to claim 11, wherein the xylitol is purified usingchromatography.